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The term glaucoma encompasses various neurodegenerative eye disorders, among which the most common is primary open-angle glaucoma (POAG). Recently, the essential role of human retinal astrocytes (HRA) in glaucoma progression has been placed in the spotlight. It has been found that placing the endoplasmic reticulum (ER) under stress and activating PERK leads to apoptosis of HRA cells, which inhibits their neuroprotective effect in the course of glaucoma. Therefore, the aim of the present study was to evaluate the effectiveness of the small-molecule PERK inhibitor LDN-0060609 in countering ER stress conditions induced in HRA cells in vitro. The activity of LDN-0060609 was studied in terms of protein and mRNA expression, cytotoxicity, genotoxicity, caspase-3 level and cell cycle progression. LDN-0060609 at 25 μM proved to be a potent inhibitor of the major PERK substrate, p-eIF2α (49% inhibition). The compound markedly decreased the expression of pro-apoptotic ER stress-related genes (ATF4, DDIT3, BAX and Bcl-2). Treatment with LDN-0060609 significantly increased cell viability, decreased genotoxicity and caspase-3 levels, and restored cell cycle distribution in HRA cells with activated ER stress conditions. These findings indicate that the small-molecule PERK inhibitor LDN-0060609 can potentially be developed into a novel anti-glaucoma agent. [ABSTRACT FROM AUTHOR]

Introduction: The newest data has reported that endoplasmic reticulum (ER) stress and PERK-dependent Unfolded Protein Response (UPR) signaling pathway may constitute a key factor in colorectal cancer (CRC) pathogenesis on the molecular level. Nowadays used anti-cancer treatment strategies are still insufficient, since patients suffer from various side effects that are directly evoked via therapeutic agents characterized by non-specific action in normal and cancer cells. Aim: Thereby, the main aim of the presented research was to analyze the effectiveness of the small-molecule PERK inhibitor NCI 12487 in an in vitro cellular model of CRC. Materials and methods: The study was performed on colorectal cancer HT-29 and normal human colon epithelial CCD 841 CoN cell lines. The cytotoxicity was measured by XTT assay, evaluation of apoptosis was performed by caspase-3 assay, whereas cell cycle analysis via the propidium iodide (PI) staining. Results: Results obtained have demonstrated that the investigated compound is selective only for HT-29 cancer cells, since at 25 µM concentration it significantly decreased HT-29 cells viability in a dose- and time-dependent manner, evoked increased caspase-3 activity and arrest in the G2/M phase of the cell cycle. Moreover, NCI 12487 compound markedly decreased HT-29 cells viability, increased caspase-3 activity and percentage of cells in sub-G0/G1, thus promoted apoptosis of cancer HT-29 cells with induced ER stress conditions. Conclusion: Thus, based on the results obtained in this study it may be concluded that small-molecule modulators of the PERK-dependent UPR signaling pathway may constitute an innovative, targeted treatment strategy against CRC. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common neurodegenerative disorder in worldwide and remains a therapeutic challenge due to the low efficacy of current treatments. Numerous studies have demonstrated the pivotal role of endoplasmic reticulum (ER) stress in PD pathogenesis. ER stress, followed by activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent branch of the unfolded protein response signaling pathway, ultimately leads to neural cell death and dopaminergic neurodegeneration in PD. Therefore, the present study evaluated the effectiveness of the small-molecule PERK inhibitor LDN-87357 in an in vitro PD model using the human neuroblastoma SH-SY5Y cell line. To assess the mRNA expression levels of the pro-apoptotic ER stress markers, the TaqMan Gene Expression Assay was performed. Cytotoxicity was assessed using a colorimetric 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay and apoptosis was assessed using a caspase-3 assay. Moreover, cell cycle progression was evaluated using flow cytometry. The results indicated that LDN-87357 treatment induced a significant decrease in ER stress markers gene expression in SH-SY5Y cells exposed to ER stress. Furthermore, LDN-87357 significantly increased viability, diminished apoptosis and restored the normal cell cycle distribution of SH-SY5Y cells after ER stress induction. Therefore, the evaluation of small-molecule PERK inhibitors, such as LDN-87357, may lead to the development of novel therapeutic strategies against PD. [ABSTRACT FROM AUTHOR]

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG. [ABSTRACT FROM AUTHOR]

Dental universal adhesives are considered an useful tool in modern dentistry as they can be used in different etching techniques, allow for simplified protocol and provide sufficient bond strength. However, there is still no consensus as to their toxicity towards pulp. Thus, the present study aimed to evaluate the cytotoxicity and genotoxicity of three universal adhesives: OptiBond Universal, Prime&Bond Universal and Adhese in an in vitro experimental model, monocyte/macrophage cell line SC (ATCC CRL-9855). The cytotoxicity was measured by means of XTT assay, whereas the genotoxicity (comet assay) was evaluated based on the percentage of DNA present in the comet tail. Furthermore, the ability of the adhesives to induce apoptosis was analyzed using flow cytometry (FC) with the FITC annexin V/propidium iodide (PI) double staining. The analysis of the cell cycle progression was performed with FC using PI staining. OptiBond Universal presented significant, while Prime&Bond Universal and Adhese Universal had minimal cytotoxicity and genotoxicity towards human SC cells. Moreover, only OptiBond Universal increased the level of apoptosis in SC cell line. None of the adhesives showed significant cell cycle arrest, as revealed by FC analysis. Due to substantial differences in toxicity in in vitro studies of dental adhesives, there is a great need for further research in order to establish more reliable test protocols allowing for standardized methodology. [ABSTRACT FROM AUTHOR]