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.

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.

[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.

[Small molecule inhibitors of PERK-dependent signaling pathway as a novel, therapeutic molecular strategy in Alzheimer's disease treatment]. / Niskoczasteczkowe inhibitory szlaku zaleznego od kinazy PERK jako nowa, molekularna strategia terapeutyczna w leczeniu choroby Alzheimera.

Nowadays more than 24 million people suffer from Alzheimer's disease (AD) that is the most common progressive cause of dementia. Molecular mechanisms of neurodegeneration in Alzheimer's disease is closely link with accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). Deposition of senile plaques is one of the main feature of Alzheimer's disease as well as is strictly correlated with impairment of cognitive abilities. The accumulation of misfolded proteins in the lumen of the ER triggers activation of the ER stress, and subsequently unfolded protein response (UPR) signaling branches, which consists of a cascade of events on the molecular level of nerve cell. That results in attenuation of global protein translation mediated by the activated Protein kinase RNA-like endoplasmic reticulum kinase (PERK) through phosphorylation of eukaryotic initiation factor 2α (eIF2α). On the contrary, prolonged ER stress contributes to preferential translation of proteins such as Activating Transcription Factor 4 (ATF4) and CCAAT-enhancer binding protein homologous protein (CHOP) engaged in apoptotic cell death. Moreover, ensues preferential translation of enzyme beta-secretase 1 (BACE1), which is an enzyme involved in deposition of senile plagues in brain tissue, that are the main cause of cognitive impairment. Recent molecular and genetic investigations present a new point of view on the therapeutic strategy for AD. Deactivation of PERK kinase via smallmolecule inhibitors has been identified as a potential therapeutic target. It is highly possible that the inhibition of PERK activity may contribute to preventing the excessive accumulation of senile plaques among the neurons and, as a result, neuronal loss and significant decline in cognitive abilities in AD.

[The role of the adaptive stress response in the pathogenesis of neurodegenerative diseases, cancer and diabetes mellitus type 2]. / Rola mechanizmu adaptacyjnej odpowiedzi na stres w patogenezie chorób neurodegeneracyjnych, nowotworowych oraz cukrzycy typu 2.

The ER (Endoplasmatic Reticulum) an intricate intracellular membrane system is responsible for many functions within cells; including folding and post-translational modifications of secretory proteins biosynthesis of ceramides, phospholipids and coordination of cell homeostasis. Perturbation of these ER processes leads to high levels unfolded and misfolded proteins within the lumen of the ER. These disturbances lead to activation of three primary receptors: PERK (Protein kinase RNA-like endoplasmic reticulum kinase), IRE1 (Inositol-Requiring-Enzyme 1) and ATF6 (Activating Transcription Factor 6). These signal transducers are responsible for inducing signalling pathways termed UPR (Unfolded Protein Response) restoring cell homeostasis. In contrast, unresolved ER stress contributes to cell death by apoptosis. Recent research allows for a conclusion that the deregulation of UPR is the main causative factor for functional cell loss and moreover, cell death by apoptosis, which is strictly linked to the pathology of human diseases to include: cancer, diabetes mellitus type 2 and neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases.

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.

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.

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.

The relationship between HDAC6, CXCR3, and SIRT1 genes expression levels with progression of primary open-angle glaucoma.

BACKGROUND: Primary open-angle glaucoma (POAG) belongs to neurodegenerative diseases. Its etiology is not fully understood. However, a lot of reports have indicated that many biochemical molecules are involved in the retinal ganglion cell damage. Therefore, the purpose of this study was to evaluate a relationship between HDAC6, CXCR3, and SIRT1 genes expression levels with the occurrence risk of POAG and its progression. MATERIALS AND METHODS: The study included 34 glaucoma patients and 32 subjects without glaucoma symptoms. RNA was isolated from peripheral blood lymphocytes. Level of mRNA expression was determined by real-time PCR method. RESULTS: Our results have shown significant association of the HDAC6 and SIRT1 expression levels with progression of POAG according to rim area (RA) value, p = 0.041; p = 0.012. Moreover, the analysis of the CXCR3 expression level showed a correlation with progression of POAG based on RA and cup disc ratio (c/d) value, p = 0.006 and p = 0.012, respectively. CONCLUSIONS: The expression level of HDAC6, CXCR3, and SIRT1 genes may be involved in the progression of POAG.

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.

Unfolded Protein Response and PERK Kinase as a New Therapeutic Target in the Pathogenesis of Alzheimer's Disease.

Recent evidence suggests that the development of Alzheimer's disease (AD) and related cognitive loss is due to mutations in the Amyloid Precursor Protein (APP) gene on chromosome 21 and increased activation of eukaryotic translation initiation factor-2α (eIF2α) phosphorylation. The high level of misfolded and unfolded proteins loading in Endoplasmic Reticulum (ER) lumen triggers ER stress and as a result Unfolded Protein Response (UPR) pathways are activated. Stress-dependent activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) leads to the significant elevation of phospho-eIF2α. That attenuates general translation and, on the other hand, promotes the preferential synthesis of Activating Transcription Factor 4 (ATF4) and secretase ß (BACE1) - a pivotal enzyme responsible for the initiation of the amyloidogenic pathway resulting in the generation of the amyloid ß (Aß) variant with high ability to form toxic senile plaques in AD brains. Moreover, excessive, long-term stress conditions may contribute to inducing neuronal death by apoptosis as a result of the overactivated expression of pro-apoptotic proteins via ATF4. These findings allow to infer that dysregulated translation, increased expression of BACE1 and ATF4, as a result of eIF2α phosphorylation, may be a major contributor to structural and functional neuronal loss resulting in memory impairment. Thus, blocking PERK-dependent eIF2α phosphorylation through specific, small-molecule PERK branch inhibitors seems to be a potential treatment strategy for AD individuals. That may contribute to the restoration of global translation rates and reduction of expression of ATF4 and BACE1. Hence, the treatment strategy can block accelerated ß -amyloidogenesis by reduction in APP cleaving via the BACE1-dependent amyloidogenic pathway.