Heat Shock Response and Heat Shock Proteins: Current Understanding and Future Opportunities in Human Diseases.

The heat shock response is an evolutionarily conserved mechanism that protects cells or organisms from the harmful effects of various stressors such as heat, chemicals toxins, UV radiation, and oxidizing agents. The heat shock response triggers the expression of a specific set of genes and proteins known as heat shock genes/proteins or molecular chaperones, including HSP100, HSP90, HSP70, HSP60, and small HSPs. Heat shock proteins (HSPs) play a crucial role in thermotolerance and aiding in protecting cells from harmful insults of stressors. HSPs are involved in essential cellular functions such as protein folding, eliminating misfolded proteins, apoptosis, and modulating cell signaling. The stress response to various environmental insults has been extensively studied in organisms from prokaryotes to higher organisms. The responses of organisms to various environmental stressors rely on the intensity and threshold of the stress stimuli, which vary among organisms and cellular contexts. Studies on heat shock proteins have primarily focused on HSP70, HSP90, HSP60, small HSPs, and ubiquitin, along with their applications in human biology. The current review highlighted a comprehensive mechanism of heat shock response and explores the function of heat shock proteins in stress management, as well as their potential as therapeutic agents and diagnostic markers for various diseases.

Neurotoxic and cardiotoxic effects of N-methyl-1-(naphthalen-2-yl)propan-2-amine (methamnetamine) and 1-phenyl-2-pyrrolidinylpentane (prolintane).

Two synthetic phenylethylamines, N-methyl-1-(naphthalen-2-yl)propan-2-amine (MNA) and 1-phenyl-2-pyrrolidinylpentane (prolintane), are being abused by people seeking hallucinogens for pleasure. These new psychotropic substances may provoke problems because there is no existing information about their toxicity and pharmacological behaviors. Therefore, we evaluated the safety of nerves and cardiovascular systems by determining toxicity after MNA and prolintane drugs administrations to mice and rat. Consequently, side effects such as increased spontaneous motion and body temperature were observed in oral administration of MNA. In addition, both substances reduced motor coordination levels. The IHC tests were conducted to see whether the immune response also shows abnormalities in brain tissue compared to the control group. It has been confirmed that the length of allograft inflammatory factor 1(IBA-1), an immune antibody known as microglia marker, has been shortened. We identified that a problem with the contact between synapses and neurons might be possibly produced. In the assessment of the cardiac toxicity harmfulness, no substances have been confirmed to be toxic to myocardial cells, but at certain concentrations, they have caused the QT prolongation, an indicator of ventricular arrhythmia. In addition, the hERG potassium channel, the biomarker of the QT prolongation, has been checked for inhibition. The results revealed that the possibility of QT prolongation through the hERG channel could not be excluded, and the two substances can be considered toxic that may cause ventricular arrhythmia. In sum, this study demonstrated that the possibility of toxicity in MNA and prolintane compounds might bring many harmful effects on nerves and hearts.

Molecular Mechanisms of Neuroprotection by Ketone Bodies and Ketogenic Diet in Cerebral Ischemia and Neurodegenerative Diseases.

Ketone bodies (KBs), such as acetoacetate and ß-hydroxybutyrate, serve as crucial alternative energy sources during glucose deficiency. KBs, generated through ketogenesis in the liver, are metabolized into acetyl-CoA in extrahepatic tissues, entering the tricarboxylic acid cycle and electron transport chain for ATP production. Reduced glucose metabolism and mitochondrial dysfunction correlate with increased neuronal death and brain damage during cerebral ischemia and neurodegeneration. Both KBs and the ketogenic diet (KD) demonstrate neuroprotective effects by orchestrating various cellular processes through metabolic and signaling functions. They enhance mitochondrial function, mitigate oxidative stress and apoptosis, and regulate epigenetic and post-translational modifications of histones and non-histone proteins. Additionally, KBs and KD contribute to reducing neuroinflammation and modulating autophagy, neurotransmission systems, and gut microbiome. This review aims to explore the current understanding of the molecular mechanisms underpinning the neuroprotective effects of KBs and KD against brain damage in cerebral ischemia and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease.

25I-NBOMe, a phenethylamine derivative, induces adverse cardiovascular effects in rodents: possible involvement of p21 (CDC42/RAC)-activated kinase 1.

Abuse of new psychoactive substances is an emerging social problem. Several phenethylamines are internationally controlled substances as they are likely to be abused and have adverse effects. Phenethylamine analog 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25I-NBOMe) has been reported as one of the most commonly abused psychoactive substance. However, the cardiotoxicity of this compound has not been extensively evaluated. Thus, in this study, we investigated the adverse cardiovascular effects of 25I-NBOMe, related to p21 (CDC42/RAC)-activated kinase 1 (PAK1). The cardiotoxicity of 25I-NBOMe was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, live/dead cytotoxicity assay, PAK1/CDC42 kinase assay, and in vivo electrocardiography (ECG). Also, we analyzed the expression level of PAK1, which is known to play key roles in the cardiovascular system. In the MTT assay, cell viability of 25I-NBOMe-treated H9c2 cells or primary cardiomyocytes of ICR mice decreased in a concentration-dependent manner. Results from the in vitro cytotoxicity assay in cardiomyocytes showed that 25I-NBOMe decreased the viability of H9c2 rat cardiomyocytes, and TC50 of 25I-NBOMe was found to be 70.4 µM. We also observed that 25I-NBOMe reduced PAK1 activity in vitro. Surface ECG measurement revealed that intravenous injection of 25I-NBOMe (doses of 1.0 and 3.0 mg/kg, corresponding to serum concentrations of 18.1 and 28.6 ng/mL, respectively) prolonged the QTc interval in SD rats. Furthermore, treatment with 25I-NBOMe downregulated the expression of PAK1 in the hearts of SD rats and H9c2 cells. In summary, our findings indicate that PAK1-related adverse effects of 25I-NBOMe can cause toxicity to cardiomyocytes and induce an abnormal ECG pattern in animals.

Honokiol inhibits hepatoma carcinoma cell migration through downregulated Cyclophilin B expression.

Hepatocellular carcinoma (HCC) is the fifth common types of cancer with poor prognosis in the world. Honokiol (HNK), a natural biphenyl compound derived from the magnolia plant, has been reported to exert anticancer effects, but its mechanism has not been elucidated exactly. In the present study, HNK treatment significantly suppressed the migration ability of HepG2 and Hep3B human hepatocellular carcinoma. The treatment reduced the expression levels of the genes associated with cell migration, such as S100A4, MMP-2, MMP-9 and Vimentin. Interestingly, treatment with HNK significantly reduced the expression level of Cyclophilin B (CypB) which stimulates cancer cell migration. However, overexpressed CypB abolished HNK-mediated suppression of cell migration, and reversed the apoptotic effects of HNK. Altogether, we concluded that the suppression of migration activities by HNK was through down-regulated CypB in HCC. These finding suggest that HNK may be a promising candidate for HCC treatment via regulation of CypB.

In vitro and in vivo estrogenic activity of triclosan.

Triclosan (TCS) is an antibacterial and antifungal agent used in many consumer products and exhibits a chemical structure similar to non-steroidal estrogen, which is known to induce endocrine disruption. Triclosan has been found in human plasma, urine, and breast milk, and the safety of TCS-containing products has been disputed. Although studies attempted to determine the estrogenic activity of TCS, no clear results have emerged. The aim of the present study was to examine estrogenic activity of TCS using an in vitro E-screen assay and an in vivo uterotrophic assay. The in vitro E-screen assay demonstrated that TCS significantly enhanced proliferation of MCF-7 breast cancer cells, although not in a concentration-dependent manner. The in vivo uterotrophic results showed no significant change in the weight of uteri obtained from TCS-administered Sprague-Dawley rats. Further, to understand the estrogenic activity attributed to TCS at the molecular level, gene-expression profiling of uterus samples was performed from both TCS- or estrogen-treated rats and the genes and cellular processes affected by TCS or estrogen were compared. Data demonstrated that both the genes and cellular processes affected by TCS or estrogen were significantly similar, indicating the possibility that TCS-mediated estrogenic activity occurred at the global transcriptome level. In conclusion, in vitro and gene-profiling results suggested that TCS exhibited estrogenic activity.

Characterization of a Parkinson's disease rat model using an upgraded paraquat exposure paradigm.

Animal models of human diseases are crucial experimental tools to investigate the mechanisms involved in disease pathogenesis and to develop new therapies. In spite of the numerous animal models currently available that reproduce several neuropathological features of Parkinson disease (PD), it is challenging to have one that consistently recapitulates human PD conditions in both motor behaviors and biochemical pathological outcomes. Given that, we have implemented a new paradigm to expose rats to a chronic low dose of paraquat (PQ), using osmotic minipumps and characterized the developed pathologic features over time. The PQ exposure paradigm used lead to a rodent model of PD depicting progressive nigrostriatal dopaminergic neurodegeneration, characterized by a 41% significant loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc), a significant decrease of 18% and 40% of dopamine levels in striatum at week 5 and 8, respectively, and a significant 1.5-fold decrease in motor performance. We observed a significant increase of microglia activation state, sustained levels of α-synucleinopathy and increased oxidative stress markers in the SNpc. In summary, this is an explorative study that allowed to characterize an improved PQ-based rat model that recapitulates cardinal features of PD and may represent an attractive tool to investigate several mechanisms underlying the various aspects of PD pathogenesis as well as for the validation of the efficacy of new therapeutic approaches that targets different mechanisms involved in PD neurodegeneration.

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells.

BACKGROUND: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell-based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. RESULTS: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. CONCLUSIONS: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129-139, 2019. © 2018 Wiley Periodicals, Inc.

Correction to: Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca2+-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells.

The original version of this article contained mistakes in figures. The western blot data for pro-caspase-3 and cleaved caspase-3 (Fig. 1d), ß-actin (Fig. 1d), PLCγ1 (Fig. 5d), and eIF2α (Fig. 7d) are incorrect. The corrected Figs. 1d, 5d, and 7d are shown below. The corrections do not influence either the validity of the published data or the conclusion described in the article.

Correction to: Apicidin induces endoplasmic reticulum stress- and mitochondrial dysfunction-associated apoptosis via phospholipase Cγ1- and Ca2+-dependent pathway in mouse Neuro-2a neuroblastoma cells.

The original version of this article contained a mistake in the figure. The Ca2 + confocal image for the 2-APB/Apicidin-120 min in Fig. 5d is incorrect. The correction does not influence either the validity of the published data or the conclusion described in the article. The corrected Fig. 5d is given below.

Correction to: Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

The original version of this article contained a mistake. The bands for HA Tag and t-ERK in Figs. 2d, 2h, 3d are incorrect. The author informs that these errors had no influence in the scientific content of the paper. The corrected figures (Figs. 2 and 3) are given below.

Fluoxetine induces apoptosis through endoplasmic reticulum stress via mitogen-activated protein kinase activation and histone hyperacetylation in SK-N-BE(2)-M17 human neuroblastoma cells.

Fluoxetine (FLX) is an antidepressant drug that belongs to the class of selective serotonin reuptake inhibitors. FLX is known to induce apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of FLX were investigated in SK-N-BE(2)-M17 human neuroblastoma cells. FLX induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid or CHOP siRNA transfection reduced FLX-induced cell death. FLX induced phosphorylation of mitogen-activated protein kinases (MAPKs) family, p38, JNK, and ERK, and an upstream kinase apoptosis signal kinase 1 (ASK1). Inhibition of MAPKs and ASK1 reduced FLX-induced cell death and CHOP expression. We then showed that FLX reduced mitochondrial membrane potential (MMP) and ER stress inhibitors as well as MAPK inhibitors ameliorated FLX-induced loss of MMP. Interestingly, FLX induced hyperacetylation of histone H3 and H4, upregulation of p300 histone acetyltransferase (HAT), and downregulation of histone deacetylases (HDACs). Treatment with a HAT inhibitor anacardic acid or p300 HAT siRNA transfection blocked FLX-induced apoptosis in SK-N-BE(2)-M17 cells. However, FLX did not induce histone acetylation and anacardic acid had no protective effect on FLX-induced cell death and CHOP expression in MYCN non-amplified SH-SY5Y human neuroblastoma and MYCN knockdowned SK-N-BE(2)-M17 cells. These findings suggest that FLX induces apoptosis in neuroblastoma through ER stress and mitochondrial dysfunction via the ASK1 and MAPK pathways and through histone hyperacetylation in a MYCN-dependent manner.

Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death.

4-Hydroxynonenal: A Superior Oxidative Biomarker Compared to Malondialdehyde and Carbonyl Content Induced by Carbon Tetrachloride in Rats.

Carbon tetrachloride (CCl4), a halogenated substance that generates free radical species during metabolism in vivo, induces hepatotoxicity, produces oxidative DNA damage, and increased levels of protein carbonyl, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In this study, Sprague-Dawley rats received single or repeated ip injections of carbon tetrachloride (CCl4), and formation and persistence of carbonyls, MDA, and 4-HNE in plasma were measured using gas chromatography-mass spectrometry. After a single injection of 500 mg/kg CCl4 the in vivo half-lives of MDA and carbonyl content were 1.5 d and 2 d, respectively, while that of 4-HNE was approximately 10 d. Treatment with CCl4 (50, 100, 500, or 1000 mg/kg) dose-dependently increased these oxidative biomarkers in blood. However, formation of protein carbonyls and MDA was less sensitive than 4-HNE to CCl4. Levels of serum glutamic oxaloacetic transaminase (SGOT) and glutamic pyruvic transaminase (SGPT) (hepatotoxicity markers) rose with CCl4 doses. After a single injection (500 mg/kg), the peak level of SGOT was observed after 8 h but SGPT after 24 h. Overall, 4-HNE was more dose-sensitive and showed greater formation subchronically than other biomarkers. Multiple ip treatments with 300 mg CCl4 /kg (d 1, 3, 6, 10, 14, and 21) demonstrated that 4-HNE formation was highest (18-fold, peak/control) and subchronic up to d 21 (last treatment day), unlike other biomarkers. Data suggest that 4-HNE, MDA, and carbonyl content may be useful oxidative biomarkers for exposure to free radical generating halogenated compounds. However, 4-HNE appears to be a more sensitive and sustainable biomarker for toxicological and risk assessments.

Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca(2+)-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells.

Licochalcone A (LicA), an estrogenic flavonoid, induces apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of LicA were investigated in HepG2 human hepatocellular carcinoma cells. LicA induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by CHOP knockdown or treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid, reduced LicA-induced cell death. LicA also induced reactive oxygen species (ROS) accumulation and the anti-oxidant N-acetylcysteine reduced LicA-induced cell death and CHOP expression. In addition, LicA increased the levels of cytosolic Ca(2+), which was blocked by 2-aminoethoxydiphenyl borate (an antagonist of inositol 1,4,5-trisphosphate receptor) and BAPTA-AM (an intracellular Ca(2+) chelator). 2-Aminoethoxydiphenyl borate and BAPTA-AM inhibited LicA-induced cell death. Interestingly, LicA induced phosphorylation of phospholipase Cγ1 (PLCγ1) and inhibition of PLCγ1 reduced cell death and ER stress. Moreover, the multi-targeted receptor tyrosine kinase inhibitors, sorafenib and sunitinib, reduced LicA-induced cell death, ER stress, and cytosolic Ca(2+) and ROS accumulation. Finally, LicA induced phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and c-Met receptor and inhibition of both receptors by co-transfection with VEGFR2 and c-Met siRNAs reversed LicA-induced cell death, Ca(2+) increase, and CHOP expression. Taken together, these findings suggest that induction of ER stress via a PLCγ1-, Ca(2+)-, and ROS-dependent pathway may be an important mechanism by which LicA induces apoptosis in HepG2 hepatocellular carcinoma cells.

p53 negatively regulates Pin1 expression under ER stress.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.