Targeting Mitochondria for Cancer Treatment.

There is an increasing accumulation of data on the exceptional importance of mitochondria in the occurrence and treatment of cancer, and in all lines of evidence for such participation, there are both energetic and non-bioenergetic functional features of mitochondria. This analytical review examines three specific features of adaptive mitochondrial changes in several malignant tumors. The first feature is characteristic of solid tumors, whose cells are forced to rebuild their energetics due to the absence of oxygen, namely, to activate the fumarate reductase pathway instead of the traditional succinate oxidase pathway that exists in aerobic conditions. For such a restructuring, the presence of a low-potential quinone is necessary, which cannot ensure the conventional conversion of succinate into fumarate but rather enables the reverse reaction, that is, the conversion of fumarate into succinate. In this scenario, complex I becomes the only generator of energy in mitochondria. The second feature is the increased proliferation in aggressive tumors of the so-called mitochondrial (peripheral) benzodiazepine receptor, also called translocator protein (TSPO) residing in the outer mitochondrial membrane, the function of which in oncogenic transformation stays mysterious. The third feature of tumor cells is the enhanced retention of certain molecules, in particular mitochondrially directed cations similar to rhodamine 123, which allows for the selective accumulation of anticancer drugs in mitochondria. These three features of mitochondria can be targets for the development of an anti-cancer strategy.

Sex-Specific Effects of Estradiol and Progesterone in Ischemic Kidney Injury.

The positive effects of female sex hormones, particularly estradiol and progesterone, have been observed in treatment of various pathologies. Acute kidney injury (AKI) is a common condition in hospitalized patients in which the molecular mechanisms of hormone action are poorly characterized. In this study, we investigated the influence of estradiol and progesterone on renal cells during ischemic injury. We performed both in vivo experiments on female and male rats and in vitro experiments on renal tubular cells (RTCs) obtained from the kidneys of intact animals of different sexes. Since mitochondria play an important role in the pathogenesis of AKI, we analyzed the properties of individual mitochondria in renal cells, including the area, roundness, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening time. We found that pre-treatment with progesterone or estradiol attenuated the severity of ischemia/reperfusion (I/R)-induced AKI in female rats, whereas in male rats, these hormones exacerbated renal dysfunction. We demonstrated that the mPTP opening time was higher in RTCs from female rats than that in those from male rats, which may be one of the reasons for the higher tolerance of females to ischemic injury. In RTCs from the kidneys of male rats, progesterone caused mitochondrial fragmentation, which can be associated with reduced cell viability. Thus, therapy with progesterone or estradiol displays quite different effects depending on sex, and could be only effective against ischemic AKI in females.

Mitochondrial Network: Electric Cable and More.

Mitochondria in a cell can unite and organize complex, extended structures that occupy the entire cellular volume, providing an equal supply with energy in the form of ATP synthesized in mitochondria. In accordance with the chemiosmotic concept, the oxidation energy of respiratory substrates is largely stored in the form of an electrical potential difference on the inner membrane of mitochondria. The theory of the functioning of extended mitochondrial structures as intracellular electrical wires suggests that mitochondria provide the fastest delivery of electrical energy through the cellular volume, followed by the use of this energy for the synthesis of ATP, thereby accelerating the process of ATP delivery compared to the rather slow diffusion of ATP in the cell. This analytical review gives the history of the cable theory, lists unsolved critical problems, describes the restructuring of the mitochondrial network and the role of oxidative stress in this process. In addition to the already proven functioning of extended mitochondrial structures as electrical cables, a number of additional functions are proposed, in particular, the hypothesis is put forth that mitochondrial networks maintain the redox potential in the cellular volume, which may vary depending on the physiological state, as a result of changes in the three-dimensional organization of the mitochondrial network (fragmentation/fission-fusion). A number of pathologies accompanied by a violation of the redox status and the participation of mitochondria in them are considered.

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

Is the Mitochondrial Membrane Potential (∆Ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the ∆Ψ Probe, Rhodamine 123.

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

Age-Related Changes in Bone-Marrow Mesenchymal Stem Cells.

The use of stem cells is part of a strategy for the treatment of a large number of diseases. However, the source of the original stem cells for use is extremely important and determines their therapeutic potential. Mesenchymal stromal cells (MSC) have proven their therapeutic effectiveness when used in a number of pathological models. However, it remains an open question whether the chronological age of the donor organism affects the effectiveness of the use of MSC. The asymmetric division of stem cells, the result of which is some residential stem cells acquiring a non-senile phenotype, means that stem cells possess an intrinsic ability to preserve juvenile characteristics, implying an absence or at least remarkable retardation of senescence in stem cells. To test whether residential MSC senesce, we evaluated the physiological changes in the MSC from old rats, with a further comparison of the neuroprotective properties of MSC from young and old animals in a model of traumatic brain injury. We found that, while the effect of administration of MSC on lesion volume was minimal, functional recovery was remarkable, with the highest effect assigned to fetal cells; the lowest effect was recorded for cells isolated from adult rats and postnatal cells, having intermediate potency. MSC from the young rats were characterized by a faster growth than adult MSC, correlating with levels of proliferating cell nuclear antigen (PCNA). However, there were no differences in respiratory activity of MSC from young and old rats, but young cells showed much higher glucose utilization than old ones. Autophagy flux was almost the same in both types of cells, but there were remarkable ultrastructural differences in old and young cells.

Cysteine Cathepsins Inhibition Affects Their Expression and Human Renal Cancer Cell Phenotype.

Renal cancer would greatly benefit from new therapeutic strategies since, in advanced stages, it is refractory to classical chemotherapeutic approaches. In this context, lysosomal protease cysteine cathepsins may represent new pharmacological targets. In renal cancer, they are characterized by a higher expression, and they were shown to play a role in its aggressiveness and spreading. Traditional studies in the field were focused on understanding the therapeutic potentialities of cysteine cathepsin inhibition, while the direct impact of such therapeutics on the expression of these enzymes was often overlooked. In this work, we engineered two fluoromethyl ketone-based peptides with inhibitory activity against cathepsins to evaluate their potential anticancer activity and impact on the lysosomal compartment in human renal cancer. Molecular modeling and biochemical assays confirmed the inhibitory properties of the peptides against cysteine cathepsin B and L. Different cell biology experiments demonstrated that the peptides could affect renal cancer cell migration and organization in colonies and spheroids, while increasing their adhesion to biological substrates. Finally, these peptide inhibitors modulated the expression of LAMP1, enhanced the expression of E-cadherin, and altered cathepsin expression. In conclusion, the inhibition of cysteine cathepsins by the peptides was beneficial in terms of cancer aggressiveness; however, they could affect the overall expression of these proteases.

Kidney Cells Regeneration: Dedifferentiation of Tubular Epithelium, Resident Stem Cells and Possible Niches for Renal Progenitors.

A kidney is an organ with relatively low basal cellular regenerative potential. However, renal cells have a pronounced ability to proliferate after injury, which undermines that the kidney cells are able to regenerate under induced conditions. The majority of studies explain yielded regeneration either by the dedifferentiation of the mature tubular epithelium or by the presence of a resident pool of progenitor cells in the kidney tissue. Whether cells responsible for the regeneration of the kidney initially have progenitor properties or if they obtain a "progenitor phenotype" during dedifferentiation after an injury, still stays the open question. The major stumbling block in resolving the issue is the lack of specific methods for distinguishing between dedifferentiated cells and resident progenitor cells. Transgenic animals, single-cell transcriptomics, and other recent approaches could be powerful tools to solve this problem. This review examines the main mechanisms of kidney regeneration: dedifferentiation of epithelial cells and activation of progenitor cells with special attention to potential niches of kidney progenitor cells. We attempted to give a detailed description of the most controversial topics in this field and ways to resolve these issues.

Effect of MSCs and MSC-Derived Extracellular Vesicles on Human Blood Coagulation.

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis.

Miro1 Enhances Mitochondria Transfer from Multipotent Mesenchymal Stem Cells (MMSC) to Neural Cells and Improves the Efficacy of Cell Recovery.

A recently discovered key role of reactive oxygen species (ROS) in mitochondrial traffic has opened a wide alley for studying the interactions between cells, including stem cells. Since its discovery in 2006, intercellular mitochondria transport has been intensively studied in different cellular models as a basis for cell therapy, since the potential of replacing malfunctioning organelles appears to be very promising. In this study, we explored the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to neural cells and analyzed its efficacy under normal conditions and upon induction of mitochondrial damage. We found that mitochondria were transferred from the MMSC to astrocytes in a more efficient manner when the astrocytes were exposed to ischemic damage associated with elevated ROS levels. Such transport of mitochondria restored the bioenergetics of the recipient cells and stimulated their proliferation. The introduction of MMSC with overexpressed Miro1 in animals that had undergone an experimental stroke led to significantly improved recovery of neurological functions. Our data suggest that mitochondrial impairment in differentiated cells can be compensated by receiving healthy mitochondria from MMSC. We demonstrate a key role of Miro1, which promotes the mitochondrial transfer from MMSC and suggest that the genetic modification of stem cells can improve the therapies for the injured brain.

Mitochondrial Aging: Is There a Mitochondrial Clock?

Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail.