Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.

Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.

New gold (III) cyanide complex TGS 121 induces ER stress, proteasome inhibition and death of Ras-hyperactivated cells.

Metal-based agents in cancer therapy, like cisplatin and its derivates, have established clinical applications but also can induce serious side effects. Thus, metallotherapeutic alternatives for platinum derivatives are developed and intensively studied. Platinum is replaced by several transition metals including gold. Especially gold (III) complexes can have the same square-planar structure and are isoelectric with platinum (II). Hence, they are developed as potential anti-cancer drugs. Thus, our group projected and developed a group of novel cyanide-based gold (III) complexes. Within this work, we aimed to characterize the safety and effectivity of one of them, TGS 121. TGS 121 in our preliminary work was selective for Ras-hyperactivated cells. Here we studied the effects of the novel complex in cancerous Ras-3 T3 and non-cancerous NIH-3 T3 cells. The complex TGS 121 turned out to be non-toxic for NIH-3 T3 cells and to induce death and alternations in Ras-hyperactivated cells. We found induction of ER stress, mitochondria swelling, proteasome inhibition, and cell cycle block. Moreover, TGS 121 inhibited cell migration and induced the accumulation of perinuclear organelles that was secondary to proteasome inhibition. Results presented in this report suggest that stable gold-cyanide TGS 121 complex is non-toxic, with a targeted mechanism of action and it is promising in anticancer drug discovery.

BDNF Spinal Overexpression after Spinal Cord Injury Partially Protects Soleus Neuromuscular Junction from Disintegration, Increasing VAChT and AChE Transcripts in Soleus but Not Tibialis Anterior Motoneurons.

After spinal cord transection (SCT) the interaction between motoneurons (MNs) and muscle is impaired, due to reorganization of the spinal network after a loss of supraspinal inputs. Rats subjected to SCT, treated with intraspinal injection of a AAV-BDNF (brain-derived neurotrophic factor) construct, partially regained the ability to walk. The central effects of this treatment have been identified, but its impact at the neuromuscular junction (NMJ) has not been characterized. Here, we compared the ability of NMJ pre- and postsynaptic machinery in the ankle extensor (Sol) and flexor (TA) muscles to respond to intraspinal AAV-BDNF after SCT. The gene expression of cholinergic molecules (VAChT, ChAT, AChE, nAChR, mAChR) was investigated in tracer-identified, microdissected MN perikarya, and in muscle fibers with the use of qPCR. In the NMJs, a distribution of VAChT, nAChR and Schwann cells was studied by immunofluorescence, and of synaptic vesicles and membrane active zones by electron microscopy. We showed partial protection of the Sol NMJs from disintegration, and upregulation of the VAChT and AChE transcripts in the Sol, but not the TA MNs after spinal enrichment with BDNF. We propose that the observed discrepancy in response to BDNF treatment is an effect of difference in the TrkB expression setting BDNF responsiveness, and of BDNF demands in Sol and TA muscles.

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity.

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.

Impact of Porcine Pancreas Decellularization Conditions on the Quality of Obtained dECM.

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy.

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting-an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells.

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis.

Mutations in GDAP1 Influence Structure and Function of the Trans-Golgi Network.

Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease that displays great genetic heterogeneity. The genes and mutations that underlie this heterogeneity have been extensively characterized by molecular genetics. However, the molecular pathogenesis of the vast majority of CMT subtypes remains terra incognita. Any attempts to perform experimental therapy for CMT disease are limited by a lack of understanding of the pathogenesis at a molecular level. In this study, we aim to identify the molecular pathways that are disturbed by mutations in the gene encoding GDAP1 using both yeast and human cell, based models of CMT-GDAP1 disease. We found that some mutations in GDAP1 led to a reduced expression of the GDAP1 protein and resulted in a selective disruption of the Golgi apparatus. These structural alterations are accompanied by functional disturbances within the Golgi. We screened over 1500 drugs that are available on the market using our yeast-based CMT-GDAP1 model. Drugs were identified that had both positive and negative effects on cell phenotypes. To the best of our knowledge, this study is the first report of the Golgi apparatus playing a role in the pathology of CMT disorders. The drugs we identified, using our yeast-based CMT-GDAP1 model, may be further used in translational research.

Three-Dimensional Segmentation and Reconstruction of Neuronal Nuclei in Confocal Microscopic Images.

The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.