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Macroautophagy (autophagy) has been a highly conserved process throughout evolution and allows cells to degrade aggregated/misfolded proteins, dysfunctional or superfluous organelles and damaged macromolecules, in order to recycle them for biosynthetic and/or energetic purposes to preserve cellular homeostasis and health. Changes in autophagy are indeed correlated with several pathological disorders such as neurodegenerative and cardiovascular diseases, infections, cancer and inflammatory diseases. Conversely, autophagy controls both apoptosis and the unfolded protein response (UPR) in the cells. Therefore, any changes in the autophagy pathway will affect both the UPR and apoptosis. Recent evidence has shown that several natural products can modulate (induce or inhibit) the autophagy pathway. Natural products may target different regulatory components of the autophagy pathway, including specific kinases or phosphatases. In this review, we evaluated ~100 natural compounds and plant species and their impact on different types of cancers via the autophagy pathway. We also discuss the impact of these compounds on the UPR and apoptosis via the autophagy pathway. A multitude of preclinical findings have shown the function of botanicals in regulating cell autophagy and its potential impact on cancer therapy; however, the number of related clinical trials to date remains low. In this regard, further pre-clinical and clinical studies are warranted to better clarify the utility of natural compounds and their modulatory effects on autophagy, as fine-tuning of autophagy could be translated into therapeutic applications for several cancers.

The angiotensin-converting enzyme 2 (ACE2) and main protease (MPro), are the putative drug candidates for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we performed 3D-QSAR pharmacophore modeling and screened 1264479 ligands gathered from Pubchem and Zinc databases. Following the calculation of the ADMET properties, molecular docking was carried out. Moreover, de novo ligand design was performed. MD simulation was then applied to survey the behavior of the ligand-protein complexes. Furthermore, MMPBSA was utilized to re-estimate the binding affinities. Then, a free energy landscape was used to find the most stable conformation of the complexes. Finally, the hybrid QM-MM method was carried out for the precise calculation of the energies. The Hypo1 pharmacophore model was selected as the best model. Our docking results indicate that the compounds ZINC12562757 and 112260215 were the best potential inhibitors of the ACE2 and MPro, respectively. Furthermore, the Evo_1 compound enjoys the highest docking energy among the designed de novo ligands. Results of RMSD, RMSF, H-bond and DSSP analyses have demonstrated that the lead compounds preserve the stability of the complexes’ conformation during the MD simulation. MMPBSA data confirmed the molecular docking results. The results of QM-MM showed that Evo_1 has a stronger potential for specific inhibition of MPro, as compared to the 112260215 compound. In conclusion, our results showed that the de novo designed Evo_1 compound has the potential to be used as a drug for the treatment of COVID-19; however, further in vitro and in vivo validations are required.

The development of novel therapeutic approaches is necessary to manage gastrointestinal cancers (GICs). Considering the effective molecular mechanisms involved in tumor growth, the therapeutic response is pivotal in this process. Autophagy is a highly conserved catabolic process that acts as a double-edged sword in tumorigenesis and tumor inhibition in a context-dependent manner. Depending on the stage of malignancy and cellular origin of the tumor, autophagy might result in cancer cell survival or death during the GICs' progression. Moreover, autophagy can prevent the progression of GIC in the early stages but leads to chemoresistance in advanced stages. Therefore, targeting specific arms of autophagy could be a promising strategy in the prevention of chemoresistance and treatment of GIC. It has been revealed that autophagy is a cytoplasmic event that is subject to transcriptional and epigenetic regulation inside the nucleus. The effect of epigenetic regulation (including DNA methylation, histone modification, and expression of non-coding RNAs (ncRNAs) in cellular fate is still not completely understood. Recent findings have indicated that epigenetic alterations can modify several genes and modulators, eventually leading to inhibition or promotion of autophagy in different cancer stages, and mediating chemoresistance or chemosensitivity. The current review focuses on the links between autophagy and epigenetics in GICs and discusses: 1) How autophagy and epigenetics are linked in GICs, by considering different epigenetic mechanisms; 2) how epigenetics may be involved in the alteration of cancer-related phenotypes, including cell proliferation, invasion, and migration; and 3) how epidrugs modulate autophagy in GICs to overcome chemoresistance.

Colorectal cancer (CRC), despite numerous therapeutic and screening attempts, still remains a major life-threatening malignancy. CRC etiology entails both genetic and environmental factors. Macroautophagy/autophagy and the unfolded protein response (UPR) are fundamental mechanisms involved in the regulation of cellular responses to environmental and genetic stresses. Both pathways are interconnected and regulate cellular responses to apoptotic stimuli. In this review, we address the epidemiology and risk factors of CRC, including genetic mutations leading to the occurrence of the disease. Next, we discuss mutations of genes related to autophagy and the UPR in CRC. Then, we discuss how autophagy and the UPR are involved in the regulation of CRC and how they associate with obesity and inflammatory responses in CRC. Finally, we provide perspectives for the modulation of autophagy and the UPR as new therapeutic options for CRC treatment.

Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor (PPAR) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPAR antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75mM palmitate reduced cell viability by 43 and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10M) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPAR-dependent and PTP1B-independent mechanisms. Copyright (c) 2014 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDYSaturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPAR-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complication and inflammatory disorders. Despite increasing scientific and clinical interest in this field, at the time of writing there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs.

Background: The leptin (LEP G2548A) and leptin receptor (LEPR Q223R) gene polymorphisms have been variably associated with type 2 diabetes (T2D) in different populations. In this study we hypothesized that these variants might be associated with T2D and related metabolic traits in an Iranian population. Methods: The LEP G2548A and LEPR Q223R genotypes were determined by PCR-RFLP in 378 normoglycemic controls and 154 T2D patients. Bonferroni correction was applied for the correction of multiple testing. Results: The A allele of the LEP G2548A polymorphism was more prevalent in females of the T2D group than the controls (p = 0.009). In a recessive model (GG+GA vs. AA), the frequency of the AA genotype was higher in female patients compared to normoglycemic subjects 34.9% vs. 19.3%, OR 2.60 (1.27 - 5.31), p = 0.009. Multivariate logistic regression analysis also showed that the AA genotype of the LEP G2548A polymorphism is an independent risk factor for T2D in females. No significant association was found between the allele and genotype frequencies of the LEPR Q223R variant with T2D in female and male groups. In addition, no significant difference in an-thropometrical and biochemical parameters was observed between the genotypes of LEP and LEPR variants in gender-specific groups in both non-diabetic and diabetic subjects. Conclusions: Our results suggest that the LEP G2548A polymorphisms might associate with T2D among Iranian female subjects, whereas the LEPR Q223R variant is not associated with T2D and its related metabolic traits in this population.

Mucolipidosis II and III are autosomal recessive disorders due to mutations in the GNPTAB and GNPTG genes encoding the alphabeta- and gamma-subunits of the GlcNAc-1-phosphotransferase, respectively. This protein has a subunit structure of alpha(2)beta(2)gamma(2) and initiates the first step of tagging lysosomal enzymes with mannose-6-phosphate (M6P). In the present study, we screened four MLII and three MLIII cell lines for mutations in GNPTAB and GNPTG. Nine novel mutations in GNPTAB and two previously reported mutations in GNPTAB and GNPTG were identified. By using anti-peptide antibodies against the alpha- and beta-subunits, we show that mutations in the gamma-subunit affected the assembly and intracellular distribution of the alpha- and beta-subunits. Furthermore, the biochemical phenotypes of MLII and MLIII fibroblasts can be corrected by transfection with wild-type cDNA expression constructs encoding the alpha/beta- and gamma-subunits of GlcNAc-1-phosphotransferase, respectively.

Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPARγ antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75 mM palmitate reduced cell viability by 43% and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10 μM) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPARγ-dependent and PTP1B-independent mechanisms.Saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPARγ-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Pediatric high-grade astrocytomas (HGAs) account for 15-20% of all pediatric central nervous system tumors. These neoplasms predominantly involve the supratentorial hemispheres or the pons--diffuse intrinsic pontine gliomas (DIPG). Assumptions that pediatric HGAs are biologically similar to adult HGAs have recently been challenged, and the development of effective therapeutic modalities for DIPG and supratentorial HGA hinges on a better understanding of their biological properties. Here, 20 pediatric HGAs (9 DIPGs and 11 supratentorial HGAs) were subject to gene expression profiling following approval by the research ethics board at our institution. Many of these tumors showed expression signatures composed of genes that promote G1/S and G2/M cell cycle progression. In particular, Aurora kinase B (AURKB) was consistently and highly overexpressed in 6/9 DIPGs and 8/11 HGAs. Array data were validated using quantitative real-time PCR and immunohistochemistry, as well as cross-validation of our data set with previously published series. Inhibition of Aurora B activity in DIPG and in pediatric HGA cell lines resulted in growth arrest accompanied by morphological changes, cell cycle aberrations, nuclear fractionation and polyploidy as well as a reduction in colony formation. Our data highlight Aurora B as a potential therapeutic target in DIPG.

Purpose Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating of pediatric malignancies and one for which no effective therapy exists. A major contributor to the failure of therapeutic trials is the assumption that biologic properties of brainstem tumors in children are identical to cerebral high-grade gliomas of adults. A better understanding of the biology of DIPG itself is needed in order to develop agents targeted more specifically to these children's disease. Herein, we address this lack of knowledge by performing the first high-resolution single nucleotide polymorphism (SNP) –based DNA microarray analysis of a series of DIPGs. Patients and Methods Eleven samples (nine postmortem and two pretreatment surgical samples), the largest series thus far examined, were hybridized to SNP arrays (250 k or 6.0). The study was approved by the research ethics board at our institution. All array findings were validated using quantitative polymerase chain reaction, fluorescence in situ hybridization, immunohistochemistry, and/or microsatellite analysis. Results Analysis of DIPG copy number alterations showed recurrent changes distinct from those of pediatric supratentorial high-grade astrocytomas. Thirty-six percent of DIPGs had gains in platelet-derived growth factor receptor alpha (PDGFRA; 4 to 18 copies) and all showed PDGFR-α expression. Low-level gains in poly (ADP-ribose) polymerase (PARP) -1 were identified in three cases. Pathway analysis revealed genes with loss of heterozygosity were enriched for DNA repair pathways. Conclusion To our knowledge, our data provides the first, comprehensive high-resolution genomic analysis of pediatric DIPG. Our findings of recurrent involvement of the PDGFR pathway as well as defects in DNA repair pathways coupled with gain of PARP-1 highlight two potential, biologically based, therapeutic targets directed specifically at this devastating disease.

The ability to use in vitro mutagenesis and transfection techniques to generate stable cell lines that highly express a mutant protein associated with a metabolic disease has been an important technique in linking genotype to clinical phenotype (Ozkara and Sandhoff, 2003). One group of metabolic diseases is GM2 gangliosidosis (AB-variant, Tay-Sachs and Sandhoff diseases). These diseases are characterized by the accumulation of GM2 ganglioside, primarily in the lysosome of neuronal tissue, leading to progressive neurodegeneration. Tay-Sachs and Sandhoff diseases are caused by defects in the HEXA gene, which encodes the α-subunit, and the HEXB gene, which encodes the β-subunit of heterodimeric N-acetyl-β-hexosaminidase (Hex) (EC 3.2.1.52), respectively (Gravel et al., 2001; Mahuran, 1999). The AB-variant is associated with defects in the GM2 activator protein, a substrate-specific cofactor for Hex A. The second major Hex isozyme in normal human tissue is Hex B, a homodimer of β-subunits. In this study, we introduced a cDNA encoding the β-subunit of human Hex (βcDNA) into three different eukaryotic expression vectors containing two different promoters, in order to establish which produces the highest expression levels of Hex B activity and β-protein in permanently transfected CHO cells. Our results indicate that the type of promoter employed can be critical in the establishment of highly expressing cell lines.

The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are approximately 60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the alpha-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant alpha-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events.

Background: The human kallikrein 6 gene (KLK6) encodes for a secreted serine protease, hK6, which is highly expressed in brain. Previous reports have associated hK6 with the pathogenesis of Alzheimer’s disease. Our objective was to develop a highly sensitive immunoassay for hK6 and use it to examine the levels of hK6 in brain tissue extracts from Alzheimer’s disease patients and control subjects. Methods: We developed antibodies against hK6 and constructed a ’sandwich’ type immunoassay. We then assessed levels of hK6 in brain extracts from normal individuals and patients with Alzheimer’s disease. Results: The hK6 assay was developed using a combination of two antibodies (a mouse monoclonal and a rabbit polyclonal). Purified recombinant hK6 was used as a calibrator. The detection limit of the assay was 0.05 g/L. The intra and inter-assay coefficient of variation was less than 6.5%. We found no detectable cross-reactivity by the homologous proteins hK2, hK3, hK8, hK10, hK11, hK13 and hK14. The hK6 concentration in human brain tissue extracts from healthy (n 24) and Alzheimer’s patients (n 55) were 10.1 1.0 and 3.39 0.26 g/g of total protein (mean SE), respectively (p 0.001). Similar differences were seen when the tissues were stratified by brain region (occipital, parietal, frontal and temporal cortex). Conclusions: We conclude that the newly developed hK6 immunoassay is suitable for quantification of hK6 protein in biologic fluids and tissue extracts. The brain of Alzheimer’s disease patients contains significantly less hK6 than the brain of nonaffected individuals. The possible connection of hK6 with the pathogenesis of Alzheimer’s disease merits further investigation. © 2002 The Canadian Society of Clinical Chemists. All rights reserved.

DIPG is one of the most devastating of pediatric malignancies and one for which no effective therapy exists. A major contributor to the failure of therapeutic trials is the assumption that biologic properties of childhood brainstem tumors are identical to adult high-grade gliomas. A better understanding of the biology of DIPG itself is needed in order to develop agents targeted more specifically to these children's disease. Here we address this lack of knowledge by performing the first high-resolution, DNA- and RNA-microarray analysis of pediatric DIPG. Nine DIPGs were prospectively collected at post-mortem following consent for research purposes. Four pre-treatment surgical samples were also retrieved from our pathology archives. All samples were hybridized to Affymetrix SNP-arrays (250k or 6.0). Nine samples were also hybridized to the HumanRef-8 v3.0 (DASL) or HumanWG-6 v3.0 expression beadchip from Illumina. Data were analyzed using the Copy Number Analysis Tool (Affymetrix) and Partek Genomics Suite using HMM and segmentation algorithm for copy number and clustering algorithm for gene expression. DASL and Whole genome beadchip from Illumina have 18,404 probes in common. Those common probes were selected in the two data sets and the gene intensities related to those probes were pulled out for each sample separately. The differentially expressed genes were then identified using one-way ANOVA. For each sample, a gene list was created containing genes having a fold change between 2 and −2. The intersection of the seven gene lists was found using a Venn diagram. In total 234 genes were found that are differentially expressed in all seven samples. The gene expression and gene copy number data were then integrated. In total 5 genes show correlation between copy number and gene expression in all seven samples. Among the 234 differentially expressed genes, Ingenuity pathway analysis identified 63 genes associated with different aspect of cancer including proliferation, metastasis, polyploidization, neoplasia, adhesion, migration, survival, acidification, attachment, cell cycle progression, differentiation, growth and invasion. Our data provides the first, comprehensive, genomic analysis of pediatric DIPG. A thorough understanding of the genetic abnormalities in DIPG is a crucial first step in the development of targeted therapies for this devastating group of tumors. This is of particular importance for DIPG given the lack of efficacy of current treatment regimens and the dismal prognosis for these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1991.

Tumors of the central nervous system constitute the most common type of solid pediatric tumors and account for the majority of mortality and morbidity in pediatric oncology. Brainstem glioma (BSG) is a type of brain tumor that arises in the medulla, pons or midbrain and accounts for approximately 10-20% of pediatric brain tumors. Diffuse gliomas of the pontine region, diffuse intrinsic pontine glioma (DIPG) are the most common type of brainstem tumor, accounting for about 58-78% of BSG and the number one cause of brain tumor related death in children with a 1-2 year survival. Despite this very little is understood about the biology of these tumors. To try to address this lack of knowledge we have undertaken genomic analysis of a series of DIPGs and compared their copy number changes to those seen in pediatric supratentorial high grade astrocytomas as well as low grade astrocytomas. Post mortem tumor and matched normal brain samples (n= 9) and surgical samples (n= 4) were collected for a series of DIPG patients. We performed high-resolution genetic analysis using whole-genome single-nucleotide polymorphism (SNP) arrays (Affymetrix 500K and 6.0). Data analysis was conducted using Partek Genome Suite and Copy Number Analysis Tool (Affymetrix). Analysis of copy number alterations returned hits in several cancer related pathways including MAPK, Wnt, prostate cancer and gliomagenesis. DIPG copy number was also compared to that of pediatric high grade (n= 20) and low grade (n= 30) astrocytomas. Unsupervised Pearson's Dissimilarity clustering resulted in 3 distinct groups. One contained 6 DIPGs; the second contained mostly LGA with a few HGA, while the third group contained mostly HGA with the remaining DIPGs clustering with this group. Although there were some similarities, DIPG are genetically distinct from supratentorial high grade and low grade astrocytomas. A better understanding of DIPG biology is needed in order to develop agents targeted more specifically towards this disease particularly given the dismal prognosis and unresponsive nature of these tumors to conventional treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2129.