Evaluation of the LDN-0060609 PERK Inhibitor as a Selective Treatment for Primary Open-Angle Glaucoma: An In Vitro Study on Human Retinal Astrocytes.

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.

The diversification of SARS-CoV-2 Omicron variants and evaluation of their detection with molecular and rapid antigen assays.

BACKGROUND: The SARS-CoV-2 pandemic saw the rapid rise, global spread, and diversification of the omicron variant in 2022. Given the overwhelming dominance of this variant globally and its diverse lineages, there is an urgent need to ensure that diagnostic assays are capable of detecting widely circulating omicron sub-lineages. STUDY DESIGN: Remnant clinical VTM samples from SARS-CoV-2 PCR confirmed infections (n = 733) collected in Wisconsin (n = 94), New York (n = 267), and South Carolina (n = 372) throughout 2022 were sequenced, classified, and tested with m2000 RealTime SARS-CoV-2, Alinity m SARS-CoV-2, ID NOW COVID-19 v2.0, BinaxNOW COVID-19 Ag Card, and Panbio COVID-19 Rapid Test Device assays. RESULTS: Sequences and lineage classifications were obtained for n = 641/733 (87.4%) samples and included delta (n = 6) and representatives from all major SARS-CoV-2 omicron variants circulating in 2022 (BA.1, BA.2, BA.3, BA.4, BA.5, BE, BF, BQ.1, and XBB). Panels of diverse omicron lineages were tested by molecular assays RealTime (n = 624), Alinity m (n = 80), and ID NOW v2.0 (n = 88) with results showing 100% detection for all samples. BinaxNOW and Panbio had sensitivities of 494/533 (92.7%) and 416/469 (88.7%), respectively for specimens with >4 log10 copies/test, consistent with expected performance for frozen specimens. Furthermore, BinaxNOW demonstrated SARS-CoV-2 detection in clinical samples 1-4 days, and up to 18 days post-symptom onset in BA.1 infected patients with >4 log10 copies/test. CONCLUSIONS: This data highlights the rise and diversification of SARS-CoV-2 omicron variants over the course of 2022 and demonstrate that each of the 5 tested assays can detect the breadth of omicron variants circulating globally.

Small-molecule inhibitors of the PERK-mediated Unfolded Protein Response signaling pathway in targeted therapy for colorectal cancer.

<b> Introduction:</b> 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. </br></br> <b>Aim:</b> 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. </br></br> <b>Materials and methods:</b> 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. </br></br> <b>Results:</b> 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. </br></br> <b>Conclusion:</b> 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.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

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.

The Structure, Activation and Signaling of IRE1 and Its Role in Determining Cell Fate.

Inositol-requiring enzyme type 1 (IRE1) is a serine/threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway, which is activated upon endoplasmic reticulum (ER) stress conditions. It is known to be capable of inducing both pro-survival and pro-apoptotic cellular responses, which are strictly related to numerous human pathologies. Among others, IRE1 activity has been confirmed to be increased in cancer, neurodegeneration, inflammatory and metabolic disorders, which are associated with an accumulation of misfolded proteins within ER lumen and the resulting ER stress conditions. Emerging evidence suggests that genetic or pharmacological modulation of IRE1 may have a significant impact on cell viability, and thus may be a promising step forward towards development of novel therapeutic strategies. In this review, we extensively describe the structural analysis of IRE1 molecule, the molecular dynamics associated with IRE1 activation, and interconnection between it and the other branches of the UPR with regard to its potential use as a therapeutic target. Detailed knowledge of the molecular characteristics of the IRE1 protein and its activation may allow the design of specific kinase or RNase modulators that may act as drug candidates.

The PERK-Dependent Molecular Mechanisms as a Novel Therapeutic Target for Neurodegenerative Diseases.

Higher prevalence of neurodegenerative diseases is strictly connected with progressive aging of the world population. Interestingly, a broad range of age-related, neurodegenerative diseases is characterized by a common pathological mechanism-accumulation of misfolded and unfolded proteins within the cells. Under certain circumstances, such protein aggregates may evoke endoplasmic reticulum (ER) stress conditions and subsequent activation of the unfolded protein response (UPR) signaling pathways via the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent manner. Under mild to moderate ER stress, UPR has a pro-adaptive role. However, severe or long-termed ER stress conditions directly evoke shift of the UPR toward its pro-apoptotic branch, which is considered to be a possible cause of neurodegeneration. To this day, there is no effective cure for Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), or prion disease. Currently available treatment approaches for these diseases are only symptomatic and cannot affect the disease progression. Treatment strategies, currently under detailed research, include inhibition of the PERK-dependent UPR signaling branches. The newest data have reported that the use of small-molecule inhibitors of the PERK-mediated signaling branches may contribute to the development of a novel, ground-breaking therapeutic approach for neurodegeneration. In this review, we critically describe all the aspects associated with such targeted therapy against neurodegenerative proteopathies.

Use of Small-molecule Inhibitory Compound of PERK-dependent Signaling Pathway as a Promising Target-based Therapy for Colorectal Cancer.

BACKGROUND: Colorectal cancer constitutes one of the most common cancer with a high mortality rate. The newest data has reported that activation of the pro-apoptotic PERK-dependent unfolded protein response signaling pathway by small-molecule inhibitors may constitute an innovative anti-cancer treatment strategy. OBJECTIVE: In the presented study, we evaluated the effectiveness of the PERK-dependent unfolded protein response signaling pathway small-molecule inhibitor 42215 both on HT-29 human colon adenocarcinoma and CCD 841 CoN normal human colon epithelial cell lines. METHODS: Cytotoxicity of the PERK inhibitor was evaluated by the resazurin-based and lactate dehydrogenase (LDH) tests. Apoptotic cell death was measured by flow cytometry using the FITCconjugated Annexin V to indicate apoptosis and propidium iodide to indicate necrosis as well as by colorimetric caspase-3 assay. The effect of tested PERK inhibitor on cell cycle progression was measured by flow cytometry using the propidium iodide staining. The level of the phosphorylated form of the eukaryotic initiation factor 2 alpha was detected by the Western blot technique. RESULTS: Obtained results showed that investigated PERK inhibitor is selective only toward cancer cells, since inhibited their viability in a dose- and time-dependent manner and induced their apoptosis and G2/M cell cycle arrest. Furthermore, 42215 PERK inhibitor evoked significant inhibition of eIF2α phosphorylation within HT-29 cancer cells. CONCLUSION: Highly-selective PERK inhibitors may provide a ground-breaking, anti-cancer treatment strategy via activation of the pro-apoptotic branch of the PERK-dependent unfolded protein response signaling pathway.

Potential therapeutic application of PERK inhibitors / Potencjalne zastosowanie terapeutyczne inhibitorów PERK.

Recent studies aimed at understanding the molecular mechanisms of human disease indicate that in the pathogenesis of many metabolic disorders, including inflammatory processes, aging of the organism, as well as cancer and neurodegenerative disorders, endoplasmic reticulum stress plays a significant role that is associated with the accumulation of misfolded proteins in the lumen of endoplasmic reticulum. In response to endoplasmic reticulum stress, the unfolded protein response pathway, that has a dualistic role, is induced. The unfolded protein response can restore endoplasmic reticulum homeostasis by degradation of unfolded proteins, inhibition of translation, and mobilization of chaperons, but it can also promote apoptosis when endoplasmic reticulum stress is prolonged. The unfolded protein response signaling pathways may be activated via three transmembrane receptors such as: PERK, IRE1 and ATF6. The most promising for development of new therapies of many human diseases, in particular cancer and neurodegeneration is PERK pathway, that inhibition shows positive therapeutic effects both in in vitro and in vivo studies.

Dual role of Endoplasmic Reticulum Stress-Mediated Unfolded Protein Response Signaling Pathway in Carcinogenesis.

Cancer constitutes a grave problem nowadays in view of the fact that it has become one of the main causes of death worldwide. Poor clinical prognosis is presumably due to cancer cells metabolism as tumor microenvironment is affected by oxidative stress. This event triggers adequate cellular response and thereby creates appropriate conditions for further cancer progression. Endoplasmic reticulum (ER) stress occurs when the balance between an ability of the ER to fold and transfer proteins and the degradation of the misfolded ones become distorted. Since ER is an organelle relatively sensitive to oxidative damage, aforementioned conditions swiftly cause the activation of the unfolded protein response (UPR) signaling pathway. The output of the UPR, depending on numerous factors, may vary and switch between the pro-survival and the pro-apoptotic branch, and hence it displays opposing effects in deciding the fate of the cancer cell. The role of UPR-related proteins in tumorigenesis, such as binding the immunoglobulin protein (BiP) and inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6) or the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), has already been specifically described so far. Nevertheless, due to the paradoxical outcomes of the UPR activation as well as gaps in current knowledge, it still needs to be further investigated. Herein we would like to elicit the actual link between neoplastic diseases and the UPR signaling pathway, considering its major branches and discussing its potential use in the development of a novel, anti-cancer, targeted therapy.

Inhibition of the PERK-Dependent Unfolded Protein Response Signaling Pathway Involved in the Pathogenesis of Alzheimer's Disease.

OBJECTIVES: There is a body of evidence that neurodegenerative disease entities are directly correlated with the perturbations on the molecular level. Hence, the ER stress-mediated Unfolded Protein Response (UPR) is activated resulting in PERK-dependent phosphorylation of the Eukaryotic initiation factor 2 (eIF2α). Thus, the levels of ATF4 and CHOP proteins are significantly increased, which subsequently switches the pro-adaptive branch of the UPR into the pro-apoptotic directly leading to neuronal loss and initiation of the neurodegenerative process. The aim of the presented study was the evaluation of the biological activity of highly specific, small-molecule inhibitors of the PERKdependent UPR signaling pathway. METHODS: The study was conducted on rat astrocytic DI TNC1 cell line. The level of p-eIF2α was measured by Western blot technique, the cytotoxicity of the investigated compound was assessed by the MTT assay and using the FITC-conjugated Annexin V (Annexin V-FITC) to indicate apoptosis and propidium iodide (PI) to indicate necrosis. The effect of tested compound on cell cycle progression was measured by flow cytometry, where the PI-labelled nuclei were analysed for DNA content. RESULTS: As a result one of the investigated compound LDN-0060609 triggers a significant inhibition of the eIF2α phosphorylation in DI TNC1 cell line. Moreover, we showed that compound LDN-0060609 is non-cytotoxic and has no effect on cell cycle progression. CONCLUSION: In conclusion, LDN-0060609 may constitute a novel, targeted treatment approach against neurodegenerative diseases, including Alzheimer's disease (AD), where pathogenesis and progression are closely associated with the overactivation of the PERK-dependent UPR signaling pathway.

[Niskoczasteczkowe inhibitory szlaku adaptacyjnej odpowiedzi na stres zaleznego od kinazy PERK jako nowatorska strategia terapeutyczna w leczeniu choroby Alzheimera]. / Small-molecule inhibitors of the PERK-dependent Unfolded Protein Response signaling pathway as a novel treatment strategy against Alzheimer's disease.

The characteristic hallmark of Alzheimer's disease (AD) are progressive changes in the brain structure and function, caused by aggregation of senile plagues, composed of improperly folded amyloid ß(Aß) protein, in the brain tissue. Recent research has suggested that causes of AD are closely associated with perturbation on the molecular level caused by the activation of the pro-apoptotic, PERKdependent Unfolded Protein Response (UPR) signaling pathway activated under Endoplasmic Reticulum (ER) stress conditions. AIM: The aims of the study were evaluation of the activity of the smallmolecule inhibitors of PERK kinase, GSK2606414 and LDN-0060609, via the analysis of the level of the phosphorylation of eIF2α as one of the main markers of the UPR signaling pathway activation as well as evaluation of the cytotoxicity of the inhibitor LDN-0060609. MATERIALS AND METHODS: The study was conducted on commercially available cell lines of wild type mouse embryotic fibroblasts 3T3 MEFs WT and with deletion of PERK gene 3T3 MEFs KO, mouse neurons CATH.a and human neuroblastoma SH-SY5Y with overexpression of amyloid precursor protein (APP). Cells were treated with commercially available inhibitor GSK2606414 or LDN-0060609, selected from the small-molecule compounds library Laboratory for Drug Discovery in Neurodegeneration, on appropriate cell culture medium with thapsigargin as an activator of Endoplasmic Reticulum (ER) stress conditions. To evaluate the level of eIF2α phosphorylation we used the Western blot technique. Detection of immune complexes was performed using the chemiluminescence. Evaluation of the LDN-0060609 compound cytotoxicity was carried out on SH-SY5Y cells using the XTT assay. RESULTS: The results of the study showed that the commercially available GSK2606414 inhibitor at a concentration of 1 µM causes >85% inhibition of the phosphorylation of eIF2α in all tested cell lines. The newly tested LDN-0060609 inhibitor showed the highest inhibitory activity at 25 µM resulting in 52% inhibition of eIF2α phosphorylation. In addition, the LDN-0060609 inhibitor did not induce a cytotoxic effect at any used concentrations and incubation times. Conclusions. It is believed that the LDN-0060609. CONCLUSIONS: It is believed that the LDN-0060609 inhibitor, that in comparison with commercially available GSK2606414 inhibitor does not evoke a cytotoxic effect, may constitute a potential factor inhibiting activation of the PERK-dependent UPR signaling pathway responsible for neurodegenerative processes in AD. Small-molecule PERK inhibitors may constitute an innovative therapeutic strategy for AD treatment.

Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells.

Menyanthes trifoliata L. has been used in traditional medicine for centuries. It exists in Asia, Europe, North America and in Morocco and is exploited as a remedy for anemia and lack of appetite. This plant shows many pharmacological properties, but its most interesting one is its anti-cancer potential. The present study examines the induction of apoptosis in grade IV glioma cells after treatment with the extracts from aerial part and root of M. trifoliata plants derived from in vitro (MtAPV and MtRV, respectively) and from soil (MtAPS and MtRS, respectively) and presents the first comparison of the biological effects of four different extracts of M. trifoliata against glioblastoma cells. The root extracts of M. trifoliata plants were found to exhibit cytotoxic effects against grade IV glioma cells, but not normal human astrocytes. HPLC analysis demonstrated the presence of various polyphenolic compounds, including sinapinic acid, ferulic acid, syringic acid and vanilic acid. Higher amount of pentacyclic triterpene (betulinic acid) was also found in MtRV extract. The growth inhibition of human grade IV glioma cells mediated by MtRV extract appears to be associated with apoptosis and G2/M phase cell cycle arrest, and altered expression of the pro- and anti-apoptotic genes (Bax, Bcl-2, Cas-3 and TP53) and proteins (Bax, Bcl-2, Cas-3 and p53), as well as decreased mitochondrial membrane potential. Our results indicate that M. trifoliata gives promising results as an anti-cancer agent for human glioblastoma cell lines. However, further research is necessary in view of its therapeutic use.

Breaking the DNA Damage Response via Serine/Threonine Kinase Inhibitors to Improve Cancer Treatment.

Multiple, both endogenous and exogenous, sources may induce DNA damage and DNA replication stress. Cells have developed DNA damage response (DDR) signaling pathways to maintain genomic stability and effectively detect and repair DNA lesions. Serine/ threonine kinases such as Ataxia-telangiectasia mutated (ATM) and Ataxia-telangiectasia and Rad3-Related (ATR) are the major regulators of DDR, since after sensing stalled DNA replication forks, DNA double- or single-strand breaks, may directly phosphorylate and activate their downstream targets, that play a key role in DNA repair, cell cycle arrest and apoptotic cell death. Interestingly, key components of DDR signaling networks may constitute an attractive target for anti-cancer therapy through two distinct potential approaches: as chemoand radiosensitizers to enhance the effectiveness of currently used genotoxic treatment or as single agents to exploit defects in DDR in cancer cells via synthetic lethal approach. Moreover, the newest data reported that serine/threonine protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is also closely associated with cancer development and progression. Thereby, utilization of small-molecule, serine/threonine kinase inhibitors may provide a novel, groundbreaking, anti-cancer treatment strategy. Currently, a range of potent, highlyselective toward ATM, ATR and PERK inhibitors has been discovered, but after foregoing study, additional investigations are necessary for their future clinical use.

Nucleotide Excision Repair Capacity and XPC and XPD Gene Polymorphism Modulate Colorectal Cancer Risk.

BACKGROUND: Colorectal cancer (CRC) is leading malignant tumors to occur mainly in industrialized countries, where it exhibits one of the highest mortality rates. Up to 80% of all CRCs characterize a chromosomal instability (CIN) phenotype. The main challenge faced by scientist is to reveal the mechanism of CIN development. An often proposed model is defects in DNA repair in terms of efficiency and genetic variations that modulate the response to stimuli from the environment. The objectives of this research were to determine whether nucleotide excision repair (NER) might affect CRC risk. MATERIALS AND METHODS: The first part of the study concerns NER efficiency. In the second part we selected 2 common single nucleotide polymorphisms within genes involved in NER (Xeroderma pigmentosum group C (XPC) Lys939Gln, Xeroderma pigmentosum group D (XPD) Lys751Gln) to determine the relation between them and CRC risk. The restriction fragment length polymorphism-polymerase chain reaction method was used for genotyping of 221 CRC patients vs. 270 cancer-free individuals. The isotopic labeling in vitro assay was used to evaluate NER capacity in lymphocytes and tissue protein extracts. RESULTS: We observed a significantly decreased level of NER capacity (P = .025) in lymphocytes delivered from CRC patients compared with healthy ones. Polymorphism screening points to higher CRC risk for the Gln939Gln genotype (P = .02) and Gln allele (P = .002) of the XPC gene. CONCLUSION: Taken together, our findings suggest a potential role for NER in CRC.

Induction of apoptosis in human glioma cell lines of various grades through the ROS-mediated mitochondrial pathway and caspase activation by Rhaponticum carthamoides transformed root extract.

The present study is the first investigation of the inhibitory effect of Rhaponticum carthamoides transformed roots (TR) extract on the proliferation of grade II and III human glioma cells. TR extract showed the cytotoxic effect and inhibited the colony formation of both glioma cell lines in dose-dependent manner. The root extract induced apoptosis by increasing of the reactive oxygen species (about threefold compared to the control cells) leading to a disruption of mitochondrial membrane potential. Additionally, the mRNA levels of the apoptotic factors such as Bax, Tp53, caspase-3, and caspase-9 were observed to increase. These results indicate that the TR extract possesses anticancer activity by inhibiting glioma cell proliferation and inducing apoptotic cell death, and may be used as a promising anticancer agent.

Inhibition of PERK-dependent pro-adaptive signaling pathway as a promising approach for cancer treatment.

Endoplasmic Reticulum (ER) is an organelle that is vital for cell growth and maintenance of homeostasis. Recent studies have reported that numerous human diseases, including cancer, are strictly connected to disruption of ER homeostasis. In order to counteract adverse intracellular conditions, cancer cells induce protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-dependent, pro-adaptive unfolded protein response (UPR) signaling branches. If ER stress is severe or prolonged, pro-adaptive signaling networks are insufficient, resulting in apoptotic cell death of cancer cells. The main aim: of the study was to evaluate the biological activity of a small-molecule PERK inhibitor GSK2606414 in two cancer cell lines - human neuroblastoma (SH-SY5Y) and human colorectal adenocarcinoma (HT-29) cell lines. We analyzed the level of phosphorylation of the eukaryotic initiation factor 2 (eIF2), which is the main substrate of PERK and a subsequent activator of UPR, which under long-term ER stress may evoke apoptotic death of cancer cells. MATERIAL AND METHODS: In the study, we utilized commercially available cell lines of human colorectal adenocarcinoma HT-29 and human neuroblastoma SH-SY5Y. Cells were exposed to the tested PERK-dependent signaling inhibitor GSK2606414 in suitable culture media with addition of thapsigargin (500 nM) to induce ER stress. To identify the protein, Western blot with specific antibodies was used. Detection of immune complexes was performed using chemiluminescence. RESULTS: We found a complete inhibition of p-eIF2α expression due to the GSK2606414 inhibitor in both cell lines, SH-SY5Y and HT-29. CONCLUSIONS: Currently available cancer treatments are insufficient and cause various side effects. It has been assumed that utilization of small-molecule inhibitors of the PERK-dependent signaling pathway, like GSK2606414, may switch the pro-adaptive branch of UPR to its pro-apoptotic branch. It is believed that the tested inhibitor GSK2606414 may become a promising treatment for many cancer types.

Antibacterial, Anti-Inflammatory, Antioxidant, and Antiproliferative Properties of Essential Oils from Hairy and Normal Roots of Leonurus sibiricus L. and Their Chemical Composition.

Essential oils obtained from the NR (normal roots) and HR (hairy roots) of the medicinal plant Leonurus sibiricus root were used in this study. The essential oil compositions were detected by GC-MS. Eighty-five components were identified in total. Seventy components were identified for NR essential oil. The major constituents in NR essential oil were ß-selinene (9.9%), selina-4,7-diene (9.7%), (E)-ß-caryophyllene (7.3%),myli-4(15)-ene (6.4%), and guaia-1(10),11-diene (5.9%). Sixty-seven components were identified in HR essential oil, the main constituents being (E)-ß-caryophyllene (22.6%), and germacrene D (19.8%). The essential oils were tested for cytotoxic effect, antimicrobial, anti-inflammatory, and antioxidant activities. Both essential oils showed activity against grade IV glioma cell lines (IC50 = 400 µg/mL), antimicrobial (MIC and MFC values of 2500 to 125 µg/mL), and anti-inflammatory (decreased level of IL-1ß, IL-6, TNF-α, and IFN-γ in LPS-stimulated cells).The essential oils exhibited moderate antioxidant activity in ABTS (EC50 = 98 and 88 µg/mL) assay. This is the first study to examine composition of the essential oils and their antimicrobial, antioxidant, antiproliferative, and anti-inflammatory activities. The results indicate that essential oils form L. sibiricus root may be used in future as an alternative to synthetic antimicrobial agents with potential application in the food and pharmaceutical industries.