Human mesenchymal stem cells increase LLC metastasis and stimulate or decelerate tumor development depending on injection method and cell amount.

Mesenchymal stem cells (MSCs) being injected into the body can stimulate or decelerate carcinogenesis. Here, the direction of influence of human placenta-derived MSCs (P-MSCs) on the Lewis lung carcinoma (LLC) tumor development and metastatic potential is investigated in C57BL/6 mice depending on the injection method. After intramuscular co-inoculation of LLC and P-MSCs (LLC + P-MSCs), the growth of primary tumor and angiogenesis are slowed down compared to the control LLC on the 15th day. This is explained by the fact of a decrease in the secretion of proangiogenic factors during in vitro co-cultivation of an equal amount of LLC and P-MSCs. When P-MSCs are intravenously (i.v.) injected in the mice with developing LLC (LLC + P-MSCs(i.v.)), the tumor growth and angiogenesis are stimulated on the 15th day. A highly activated secretion of proangiogenic factors by P-MSCs in a similar in vitro model can explain this. In both the models compared to the control on the 23rd day, there is no significant difference in the tumor growth, while angiogenesis remains correspondingly decelerated or stimulated. However, in both the models, the total volume and number of lung metastases constantly increase compared to the control: it is mainly due to small-size metastases for LLC + P-MSCs(i.v.) and larger ones for LLC + P-MSCs. The increase in the rate of LLC cell dissemination after the injection of P-MSCs is explained by the disordered polyploidy and chromosomal instability, leading to an increase in migration and invasion of cancer cells. After LLC + P-MSCs co-inoculation, the tumor cell karyotype has the most complex and heterogeneous chromosomal structure. These findings indicate a bidirectional effect of P-MSCs on the growth of LLC in the early periods after injection, depending on the injection method, and, correspondingly, the number of contacting cells. However, regardless of the injection method, P-MSCs are shown to increase LLC aggressiveness related to cancer-associated angiogenesis and metastasis activation in the long term.

Towards in vivo photomediated delivery of anticancer peptides: Insights from pharmacokinetic and -dynamic data.

An in vivo study of a photoswitchable cytotoxic peptide LMB040 has been undertaken on a chemically induced hepatocellular carcinoma model in immunocompetent rats. We analysed the pharmacokinetic profile of the less toxic photoform ("ring-closed" dithienylethene) of the compound in tumors, plasma, and healthy liver. Accordingly, the peptide can reach a tumor concentration sufficiently high to exert a cytotoxic effect upon photoconversion into the more active ("ring-open") photoform. Tissue morphology, histology, redox state of the liver, and hepatic biochemical parameters in blood serum were analysed upon treatment with (i) the less active photoform, (ii) the in vivo light-activated alternative photoform, and (iii) compared with a reference chemotherapeutic 5-fluorouracil. We found that application of the less toxic form followed by a delayed in vivo photoconversion into the more toxic ring-open form of LMB040 led to a higher overall survival of the animals, and signs of enhanced immune response were observed compared to the untreated animals.

Near-infrared light reduces ß-amyloid-stimulated microglial toxicity and enhances survival of neurons: mechanisms of light therapy for Alzheimer's disease.

BACKGROUND: Low-intensity light can decelerate neurodegenerative disease progression and reduce amyloid ß (Aß) levels in the cortex, though the cellular and molecular mechanisms by which photobiomodulation (PBM) protects against neurodegeneration are still in the early stages. Microglia cells play a key role in the pathology of Alzheimer's disease by causing chronic inflammation. We present new results concerning the PBM of both oxidative stress and microglia metabolism associated with the activation of metabolic processes by 808 nm near-infrared light. METHODS: The studies were carried out using healthy male mice to obtain the microglial cell suspension from the hippocampus. Oligomeric ß-amyloid (1-42) was prepared and used to treat microglia cells. Light irradiation of cells was performed using diode lasers emitting at 808 nm (30 mW/cm2 for 5 min, resulting in a dose of 10 J/cm2). Mitochondrial membrane potential, ROS level studies, cell viability, apoptosis, and necrosis assays were performed using epifluorescence microscopy. Phagocytosis, nitric oxide and H2O2 production, arginase, and glucose 6-phosphate dehydrogenase activities were measured using standard assays. Cytokines, glucose, lactate, and ATP were measurements with ELISA. As our data were normally distributed, two-way ANOVA test was used. RESULTS: The light induces a metabolic shift from glycolysis to mitochondrial activity in pro-inflammatory microglia affected by oligomeric Aß. Thereby, the level of anti-inflammatory microglia increases. This process is accompanied by a decrease in pro-inflammatory cytokines and an activation of phagocytosis. Light exposure decreases the Aß-induced activity of glucose-6-phosphate dehydrogenase, an enzyme that regulates the rate of the pentose phosphate pathway, which activates nicotinamide adenine dinucleotide phosphate oxidases to further produce ROS. During co-cultivation of neurons with microglia, light prevents the death of neurons, which is caused by ROS produced by Aß-altered microglia. CONCLUSIONS: These original data clarify reasons for how PBM protects against neurodegeneration and support the use of light for therapeutic research in the treatment of Alzheimer's disease.

Red and near infrared light-stimulated angiogenesis mediated via Ca2+ influx, VEGF production and NO synthesis in endothelial cells in macrophage or malignant environments.

Irradiation with red or near-infrared (NIR) light in low level light therapy (LLLT) is found to stimulate cellular processes and bioenergetics, resulting in enhanced wound healing, pain control, neurodegenerative diseases treatment, etc. During light irradiation of tissues and organs, different cells are affected, though the connection between photostimulation of cells and their environmental conditions remains poorly understood. In this report, red/NIR light-stimulated angiogenesis is investigated using endothelial cells in vitro, with a focus on the capillary-like structure (CLS) formation and the respective biochemical processes in cells under conditions proximate to a healthy or malignant environment, which strongly defines angiogenesis. To model environmental conditions for endotheliocytes in vitro, the cell culture environment was supplemented by an augmented conditioned medium from macrophages or cancer cells. The biochemical processes in endothelial cell cultures were investigated with and without irradiation by red (650 nm) and near-infrared (808 nm) laser diodes and under normoxia or hypoxia conditions. A light-stimulated angiogenesis has been found, with a more efficient stimulation by 650 nm light compared to 808 nm light. It was shown that the irradiation with light promoted extracellular Ca2+ influx, fostered cell cycle progression, proliferation and NO generation in endothelial cells, and caused an increase in vascular endothelial growth factor (VEGF) production by endothelial cells and M2 macrophages under hypoxia conditions. The activation of VEGF production by macrophages was found to be associated with an increase in the number of M2 macrophages after light irradiation under hypoxia conditions. Thus, a new pathway of an activation of the endothelial cell metabolism, which is related with the extracellular Ca2+ influx after light irradiation, has been revealed. STATEMENT OF SIGNIFICANCE: Red/NIR light-stimulated angiogenesis has been studied using endothelial cells in vitro, with focus on CLS formation and the respective biochemical processes in cell models proximate to a healthy or malignant environment. A light-stimulated angiogenesis has been found, stimulated via extracellular Ca2+ influx, cell cycle progression, proliferation and NO generation, VEGF production increase by endothelial cells under hypoxia conditions.

NMDA receptor expression during cell transformation process at early stages of liver cancer in rodent models.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, which is not sensitive to radiotherapy and chemotherapy and very often experiences postoperative relapse. In this regard, effective screening of liver cancer is considered as the most important and urgent task. The aim of our study was to determine whether N-methyl-D-aspartate receptor (NMDAR) and, in particular, its subunits, can serve as biomarkers to distinguish the precancerous liver at early stages of liver fibrosis. We assessed the development of HCC after 10, 15, and 22 wk using a HCC rat model. The expression of NMDAR subunits was monitored at different stages of HCC by means of immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting, and direct bisulfite sequencing. NMDAR subunits were not found in healthy liver tissues. In contrast, NMDAR subunits, in particular NR1 and NR2B, appeared at the stage of severe liver fibrosis (precancerous liver disease) in rats and were expressed during the development of HCC in rats and mice. Using the direct bisulfite sequencing, we detected that increased expression of NMDAR directly correlated with the demethylation of CpG islands in the promoter region of genes encoding receptor subunits. The obtained results confirmed that NMDAR subunits can serve as new biomarkers of precancerous liver disease, severe fibrosis, and its progression towards HCC.NEW & NOTEWORTHY We have shown NMDAR expression in cell transformation process at early stages of cancer, specifically HCC. The aim of our study was to define the disease stages from precancerous liver disease towards liver cancer progression when NMDAR subunits were expressed/detected. A fibrosis/HCC rat model, immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting was used. The dynamics of appearance of NMDAR subunits, their expression and methylation status during the development of HCC were shown and discussed.

Macrophages Modulated by Red/NIR Light: Phagocytosis, Cytokines, Mitochondrial Activity, Ca2+ Influx, Membrane Depolarization and Viability.

Low-level light therapy (LLLT) is emerging as a promising therapeutic approach to modulate the biochemical and molecular processes within living cells. LLLT is known to produce local and systemic effects; therefore, immune cells in local tissues or in the circulation are affected by light. However, this specific effect remains weakly explored. In this study, the effect of red (650 nm) and NIR (808 nm) light on phagocytosis (respiratory burst), cytokine expression, mitochondrial activity, ROS generation, Ca2+ influx and membrane depolarization in macrophages in vitro is investigated. Both the phagocytic capacity and adhesion of macrophages strongly (~2.5 times) increased in the first hours after exposure to light in a dose-dependent manner. The light-evoked upregulation of phagocytosis is found to be less efficient than the maximal pharmacologically induced enhancement of ~3.2 times. Also, red/NIR light reduces the production of pro-inflammatory cytokines and activates the secretion of anti-inflammatory cytokines by several times in activated macrophages. At the same time, the viability shows a biphasic dose response: it increases after irradiation with lower doses (0.3-1 J cm-2 ) and decreases after treatment with higher doses (18-30 J cm-2 ), which is apparently associated with the upregulation of ROS generation, followed by an increase in the mitochondrial activity.

Effect of Cytokinin-Containing Extracts from Some Medicinal Mushroom Mycelia on HepG2 Cells In Vitro.

Endogenous cytokinins in mycelia of medicinal mushrooms Hericium coralloides and Fomitopsis officinalis grown in vitro were identified using high-performance liquid chromatography coupled with mass spectrometry. High amounts of zeatin-type cytokinins and isopentenyladenine were found. The qualitative composition and quantitative content of cytokinins were species-specific traits of mushrooms. Optical microscopy was used to perform a comparison analysis of the influence of crude extracts and purified cytokinin fractions from both species' mycelial biomass on HepG2 tumor cell growth in vitro and morphology. The results showed that purified cytokinin fractions from H. coralloides and F. officinalis mycelia demonstrated a cytotoxic effect on HepG2 cells, unlike crude extracts. Under the influence of all mushroom extracts, similar patterns of changes in HepG2 cell morphology were observed, but they were more pronounced for H. coralloides compared with F. officinalis. Purified fractions of both mushroom species caused an increased level of apoptosis compared to crude extracts. Some increase in glucose uptake by cultured cells was found in all investigated samples wherein the influence of H. coralloides extracts was approximately twice the effect of the corresponding F. officinalis extracts. The data obtained confirm the assumption that cytokinins are involved in the expression of therapeutic effects of medicinal mushrooms and indicate the need to take into consideration the methods of cytokinin extraction when preparing pharmacologically active drugs based on fungal raw materials. Thus, extracts from H. coralloides and F. officinalis mycelial biomass are promising in the search for anticancer agents.

Red and near-infrared light induces intracellular Ca2+ flux via the activation of glutamate N-methyl-D-aspartate receptors.

Red and near-infrared (NIR) light effect on Ca2+ ions flux through the influence on N-methyl-D-aspartate receptors (NMDARs) and their functioning in HeLa cells was studied in vitro. Cells were irradiated by 650 and 808 nm laser light at different power densities and doses and the obtained effect was compared with that caused by the pharmacological agents. The laser light was found to elevate Ca2+ influx into cell cytoplasm in a dose-dependent manner without changes of the NMDAR functioning. Furthermore, the light of both wavelengths demonstrated the ability to elevate Ca2+ influx under the pharmacological blockade of NMDARs and also might partially abolish the blockade enhancing Ca2+ influx after selective stimulation of the receptors with NMDA. Simultaneously, the light at moderate doses demonstrated a photobiostimulating effect on cells. Based on our experiments and data reported in the literature, we suggest that the low-power visible and NIR light can instigate a cell membrane depolarization via nonthermal activation, resulting in the fast induction of Ca2+ influx into cells. The obtained results also demonstrate that NIR light can be used for nonthermal and nonpharmacological stimulation of NMDARs in cancer cells.

Structure-Activity Relationships of Photoswitchable Diarylethene-Based ß-Hairpin Peptides as Membranolytic Antimicrobial and Anticancer Agents.

Five series (28 structures) of photoswitchable ß-hairpin peptides were synthesized based on the cyclic scaffold of the natural antibiotic gramicidin S. Cell-type selectivity was compared for all activated (diarylethene "ring-open") and deactivated ("ring-closed") forms in terms of antibacterial activity (MIC against Escherichia coli and Bacillus subtilis), anticancer activity (IC50 against HeLa cell line), and hemolytic cytotoxicity (HC50 against human erythrocytes). Correlations between the conformational plasticity of the peptides, their hydrophobicity, and their bioactivity were also analyzed. Considerable improvements in selectivity were achieved compared to the reference compound. We found a dissociation of the anticancer activity from hemolysis. Phototherapeutic indices (PTI), HC50(closed)/MIC(open) and HC50(closed)/IC50(open), were introduced for the peptides as safety criteria. The highest PTI for HeLa-selective toxicity were observed among analogues containing hydroxyleucine on the hydrophobic face. For one compound, high PTIs were demonstrated across a range of different cancer cell lines, including a doxorubicin-resistant one.

Novel Hybrid Compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide with Antimetastatic and Cytotoxic Action: Synthesis and Anticancer Screening.

BACKGROUND: One of the most promising strategies to develop multi-targeted anticancer therapeutics is to introduce to the structure of a potential drug two or more pharmacophores (functional groups or structural fragments), which have antiproliferative, proapoptotic or antimetastatic properties acting via different mechanisms. OBJECTIVE: To design, synthesize and perform screening of a novel hybrid anticancer compound. METHOD: A novel hybrid compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide, combining butanehydroxamate and styrenesulfonamide moieties, was designed, synthesized and investigated as a potent antimetastatic and antiproliferative agent. The structure and purity of the synthesized compound were confirmed by 1H NMR, 13C NMR, LC/MS spectroscopy and elemental analysis. The compound was screened for the anticancer activity in vitro against HeLa and in vivo against Lewis lung carcinoma tumor, using an antitumor metalloenzyme inhibitor GM6001 (Ilomastat, Galardin) and Pifithrin-µ as control anticancer agents. RESULTS: It was found that the application of our compound resulted in a high fraction of apoptotic cells in the cell population, along with disruption in the cell cycle profile manifested as arrest of proliferative phases. Furthermore, changes of the morphological properties (i.e., an enhancement of adhesive properties and reduction of the nuclear-to-cytoplasm ratio) were found. The in vivo screening revealed that the compound significantly inhibited the metastasizing process that was manifested by a reduction in the number and volume of metastases. CONCLUSIONS: The obtained results demonstrate that our compound can serve as a base for further structure optimization in order to design new highly-effective antimetastatic and antitumor agents.

Direct Photocontrol of Peptidomimetics: An Alternative to Oxygen-Dependent Photodynamic Cancer Therapy.

Conventional photodynamic treatment strategies are based on the principle of activating molecular oxygen in situ by light, mediated by a photosensitizer, which leads to the generation of reactive oxygen species and thereby causes cell death. A diarylethene-derived peptidomimetic is presented that is suitable for photodynamic cancer therapy without any involvement of oxygen. This light-sensitive molecule is not a mediator but is itself the cytotoxic agent. As a derivative of the cyclic amphiphilic peptide gramicidin S, the peptidomimetic exists in two thermally stable photoforms that are interconvertible by light of different wavelengths. The isomer generated by visible light shows much stronger toxicity against tumor cells than the UV-generated isomer. First in vivo applications are demonstrated on a tumor animal model to illustrate how the peptidomimetic can be administered in the less toxic form and then activated locally in a solid tumor by visible light.