Effect of photosensitizers photosens, photodithazine and hypericin on glioma cells and primary neuronal cultures : a comparative analysis

The aim of the study was to compare the effect of photosensitizers photosens, photodithazine, and hypericin on primary brain cell cultures, and assess their toxic effect on tumor and normal nervous cells in order to choose the optimal photodynamic agent for glioma therapy. Materials and Methods. The cytotoxicity of photosens (NIOPIK, Russia), photodithazine (Veta-grand, Russia) and hypericin (Merck KGaA; Sigma-Aldrich, Germany) was assessed on primary brain cell cultures obtained from C57BL/6 mice (gestation day 18). On day 14 of cultivation, the tested photosensitizers were added to a culture medium at concentrations of 0.1, 1, 10, 50, and 100 mu M. Then the cultures were placed in a CO2-incubator in the dark. The viability of primary neuronal cultures was estimated on days 3 and 7 after photosensitizer application. Using confocal microscopy, we analyzed the rate of entry and subcellular localization of the tested agents in the primary neuronal cells. Statistical analysis was performed in SigmaPlot 11.0 (Systat Software Inc., USA) using ANOVA. Results. We analyzed the absorption and fluorescence spectra of the tested photosensitizers. Photosens and photodithazine showed the presence of absorption maximum in short- and long-wave spectral ranges. Hypericin was characterized by a complex spectrum with many peaks in both blue-violet and orange-red spectral ranges. Cell viability analysis revealed that high concentrations of photosensitizers caused a pronounced toxic effect on nervous cells. The most marked effect was shown for photodithazine. Photosens exhibited the lowest accumulation rate in primary neuronal cells. Photosens and hypericin were found to have a high phototoxic effect on glioma, and demonstrated low dark toxicity for normal brain cells. Conclusion. The photosensitizers hypericin and photosens are the least toxic for nervous tissue, though effectively penetrating in tumor cells. These properties enable to consider them as prospective photodynamic agents for clinic

An emerging role for nanomaterials in increasing immunogenicity of cancer cell death

In the last decade, it has become clear that anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). ICD is an umbrella term covering several cell death modalities, including apoptosis and necroptosis. In general, ICD is characterized by the emission of damage-associated molecular patterns (DAMPs) and/or cytokines/chemokines, leading to the induction of strong anti-tumor immune responses. In experimental cancer therapy, new observations indicate that the immunogenicity of dying cancer cells can be improved by the use of biomaterials. In this review, after a brief overview of the basic principles of the concept of ICD and discussion of the potential use of DAMPs as biomarkers of therapy efficacy, we discuss an emerging role of nanomaterials as a promising strategy to modulate the immunogenicity of cancer cell death. We address how nanocarriers can be used to increase the immunogenicity of ICD and then turn our attention to their dual action. Nanocarriers can be used to increase the immunogenicity of dying cancer cells and to reduce the side effects of chemotherapy. Future studies will show whether biomaterials are truly an optimal strategy to modulate the immunogenicity of dying cancer cells and will provide the insights needed for the development of novel treatment strategies for cancer

Necroptosis in CNS diseases: Focus on astrocytes

In the last few years, necroptosis, a recently described type of cell death, has been reported to play an important role in the development of various brain pathologies. Necroptosis is a cell death mechanism that has morphological characteristics similar to necrosis but is mediated by fundamentally different molecular pathways. Necroptosis is initiated by signaling through the interaction of RIP1/RIP3/MLKL proteins (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein). RIPK1 kinase is usually inactive under physiological conditions. It is activated by stimulation of death receptors (TNFR1, TNFR2, TLR3, and 4, Fas-ligand) by external signals. Phosphorylation of RIPK1 results in the formation of its complex with death receptors. Further, complexes with the second member of the RIP3 and MLKL cascade appear, and the necroptosome is formed. There is enough evidence that necroptosis plays an important role in the pathogenesis of brain ischemia and neurodegenerative diseases. In recent years, a point of view that both neurons and glial cells can play a key role in the development of the central nervous system (CNS) pathologies finds more and more confirmation. Astrocytes play complex roles during neurodegeneration and ischemic brain damage initiating both impair and protective processes. However, the cellular and molecular mechanisms that induce pathogenic activity of astrocytes remain veiled. In this review, we consider these processes in terms of the initiation of necroptosis. On the other hand, it is important to remember that like other types of programmed cell death, necroptosis plays an important role for the organism, as it induces a strong immune response and is involved in the control of cancerogenesis. In this review, we provide an overview of the complex role of necroptosis as an important pathogenetic component of neuronal and astrocyte death in neurodegenerative diseases, epileptogenesis, and ischemic brain damage

Age-related DNA methylation changes are sex-specific: a comprehensive assessment

The existence of a sex gap in human health and longevity has been widely documented. Autosomal DNA methylation differences between males and females have been reported, but so far few studies have investigated if DNA methylation is differently affected by aging in males and females. We performed a meta-analysis of 4 large whole blood datasets, comparing 4 aspects of epigenetic age-dependent remodeling between the two sexes: differential methylation, variability, epimutations and entropy. We reported that a large fraction (43%) of sex-associated probes undergoes age-associated DNA methylation changes, and that a limited number of probes show age-by-sex interaction. We experimentally validated 2 regions mapping in FIGN and PRR4 genes and showed sex-specific deviations of their methylation patterns in models of decelerated (centenarians) and accelerated (Down syndrome) aging. While we did not find sex differences in the age-associated increase in epimutations and entropy, we showed that the number of probes having an age-related increase in methylation variability is 15 times higher in males compared to females. Our results can offer new epigenetic tools to study the interaction between aging and sex and can pave the way to the identification of molecular triggers of sex differences in longevity and age-related diseases prevalence

Immunogenic cell death induced by a new photodynamic therapy based on photosens and photodithazine

Background: Anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). Therefore, when developing new treatment strategies, it is extremely important to choose methods that induce ICD and thereby activate anti-tumor immune response leading to the most effective destruction of tumor cells. The aim of this work was to analyze whether the clinically widely used photosensitizers, photosens (PS) and photodithazine (PD), can induce ICD when used in photodynamic therapy (PDT). Methods: Cell death in murine glioma GL261 or fibrosarcoma MCA205 cells was induced by PS- or PD-PDT and cell death was analyzed by MTT or flow cytometry. Intracellular distribution of PS and PD was studied by using the laser scanning microscope. Calreticulin exposure and HMGB1 and ATP release were detected by flow cytometry, ELISA and luminescence assay, respectively. Immunogenicity in vitro was analyzed by co-culturing of dying cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and maturation (CD11c(+)CD86(+), CD11c(+)CD40(+)) of BMDCs and production of IL-6 in the supernatant were measured. In vivo immunogenicity was analyzed in mouse tumor prophylactic vaccination model. Results: We determined the optimal concentrations of the photosensitizers and found that at a light dose of 20 J/cm(2) (lambda ex 615-635 nm) both PS and PD efficiently induced cell death in glioma GL261 and fibrosarcoma MCA205 cells. We demonstrate that PS localized predominantly in the lysosomes and that the cell death induced by PS-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) and by ferrostatin-1 and DFO (ferroptosis inhibitors), but not by the necroptosis inhibitor necrostatin-1 s. By contrast, PD accumulated in the endoplasmic reticulum and Golgi apparatus, and the cell death induced by PD-PDT was inhibited only by z-VAD-fmk. Dying cancer cells induced by PS-PDT or PD-PDT emit calreticulin, HMGB1 and ATP and they were efficiently engulfed by BMDCs, which then matured, became activated and produced IL-6. Using dying cancer cells induced by PS-PDT or PD-PDT, we demonstrate the efficient vaccination potential of ICD in vivo. Conclusions: Altogether, these results identify PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy

Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

Background: Immunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown. Methods: Ferroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c(+)CD86(+), CD11c(+)CD40(+), CD11c(+)MHCII(+), IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2(-/-)) mice was used to analyze ferroptosis immunogenicity. Results: Ferroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors' analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2(-/-) mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells. Conclusions: These results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and opens the door for the development of new strategies in cancer immunotherapy

Effect of novel porphyrazine photosensitizers on normal and tumor brain cells

Photodynamic therapy (PDT) is a clinically approved procedure for targeting tumor cells. Though several different photosensitizers have been developed, there is still much demand for novel photosensitizers with improved properties. In this study we aim to characterize the accumulation, localization and dark cytotoxicity of the novel photosensitizers developed in-house derivatives of porphyrazines (pz I-IV) in primary murine neuronal cells, as well as to identify the concentrations at which pz still effectively induces death in glioma cells yet is nontoxic to nontransformed cells. The study shows that incubation of primary neuronal and glioma cells with pz I-IV leads to their accumulation in both types of cells, but their rates of internalization, subcellular localization and dark toxicity differ significantly. Pz II was the most promising photosensitizer. It efficiently killed glioma cells while remaining nontoxic to primary neuronal cells. This opens up the possibility of evaluating pz II for experimental PDT for glioma

Brain-Derived Neurotrophic Factor (BDNF) Preserves the Functional Integrity of Neural Networks in the β-Amyloidopathy Model in vitro.

peer reviewedAlzheimer's disease (AD) is a widespread chronic neurodegenerative pathology characterized by synaptic dysfunction, partial neuronal death, cognitive decline and memory impairments. The major hallmarks of AD are extracellular senile amyloid plaques formed by various types of amyloid proteins (Aβ) and the formation and accumulation of intracellular neurofibrillary tangles. However, there is a lack of relevant experimental models for studying changes in neural network activity, the features of intercellular signaling or the effects of drugs on the functional activity of nervous cells during AD development. In this work, we examined two experimental models of amyloidopathy using primary hippocampal cultures. The first model involves the embryonic brains of 5xFAD mice; the second uses chronic application of amyloid beta 1-42 (Aβ1-42). The model based on primary hippocampal cells obtained from 5xFAD mice demonstrated changes in spontaneous network calcium activity characterized by a decrease in the number of cells exhibiting Ca(2+) activity, a decrease in the number of Ca(2+) oscillations and an increase in the duration of Ca(2+) events from day 21 of culture development in vitro. Chronic application of Aβ1-42 resulted in the rapid establishment of significant neurodegenerative changes in primary hippocampal cultures, leading to marked impairments in neural network calcium activity and increased cell death. Using this model and multielectrode arrays, we studied the influence of amyloidopathy on spontaneous bioelectrical neural network activity in primary hippocampal cultures. It was shown that chronic Aβ application decreased the number of network bursts and spikes in a burst. The spatial structure of neural networks was also disturbed that characterized by reduction in both the number of key network elements (hubs) and connections between network elements. Moreover, application of brain-derived neurotrophic factor (BDNF) recombinant protein and BDNF hyperexpression by an adeno-associated virus vector partially prevented these amyloidopathy-induced neurodegenerative phenomena. BDNF maintained cell viability and spontaneous bioelectrical and calcium network activity in primary hippocampal cultures

The Influence of Neurotrophic Factors BDNF and GDNF Overexpression on the Functional State of Mice and Their Adaptation to Audiogenic Seizures.

peer reviewedThe high prevalence of diagnosed cases of severe neurological disorders, a significant proportion of which are epilepsy, contributes to a high level of mortality and disability in the population. Neurotrophic factors BDNF and GNDF are considered promising agents aimed at increasing the central nervous system's adaptive potential for the development of the epileptiform activity. Despite the pronounced neuroprotective and anticonvulsant potential, an appropriate way to stimulate these endogenous signaling molecules with minimal risk of side effects remains an open question. Herein, we assessed the safety of gene therapy using original adeno-associated viral constructs carrying the genes of neurotrophic factors BDNF and GDNF in the early postnatal period of development of experimental animals. The intraventricular injection of AAV-Syn-BDNF-eGFP and AAV-Syn-GDNF-eGFP viral constructs into newborn mice was found to provide persistent overexpression of target genes in the hippocampus and cerebral cortex in vivo for four weeks after injection. The application of viral constructs has a multidirectional effect on the weight and body length characteristics of mice in the early postnatal period; however, it ensures the animals' resistance to the development of seizure activity under audiogenic stimulation in the late postnatal period and preserves basic behavioral reactions, emotional status, as well as the mnestic and cognitive abilities of mice after simulated stress. Our results demonstrated the safety of using the AAV-Syn-BDNF-eGFP and AAV-Syn-GDNF-eGFP viral constructs in vivo, which indicates the expediency of further testing the constructs as therapeutic anticonvulsants