Glycogenin is Dispensable for Glycogen Synthesis in Human Muscle, and Glycogenin Deficiency Causes Polyglucosan Storage

Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. OBJECTIVE: The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS: Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. RESULTS: Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS: Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy

Lowered expression of tumor suppressor candidate MYO1C stimulates cell proliferation, suppresses cell adhesion and activates AKT

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of wellstratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition.Royal Physiographic Society in Lund (Nilsson-Ehle Foundation) with grant numbers 30928, 32705 and 36388: KV. Wilhelm and Martina Lundgren Foundation: KV, AB. Assar Gabrielsson Research Foundation for Clinical Cancer Research with grant numbers FB11-15, FB12-26, FB13-05, FB14-46 and FB15-45: KV. Sahlgrenska University Hospital Foundation with grant number 8181: KV. The Knowledge Foundation with grant number HOÈ G12, 20120311: AB.http://www.plosone.orgam2016Physiolog

Cellular, Molecular and Functional Characterization of the Tumor Suppressor Candidate MYO1C

Tumor suppressor genes play a role as a growth regulator and a gatekeeper of a cell. Their inactivation is often detected in malignant tumors. Identification of novel tumor suppressor gene candidates may help to further understand tumorigenesis and aid in the discovery of a new treatment leading toward cure of cancer. This PhD research project aimed to understand functional significance of a novel tumor suppressor gene candidate, myosin IC (MYO1C) and to identify potential interaction(s) of the MYO1C protein with key components of the signaling pathways involving in cancer development. In an experimental rat model for endometrial carcinoma (EC), detailed molecular genetic analysis of a candidate tumor suppressor region located distal to the tumor protein 53 (Tp53) suggested the myosin IC gene (Myo1c) as the best potential target for deletion of the genetic material. The question arising was whether and how MYO1C could function as a tumor suppressor gene. By using qPCR, Western blot or immunohistochemistry analyses, we examined MYO1C protein level in panels of well-stratified human colorectal cancer (CRC) and EC respectively. We found that MYO1C was significantly down-regulated in these cancer materials and that for the EC panel, the observed down-regulation of MYO1C correlated with tumor stage, where tumors at more advanced stages had less expression of MYO1C. In cell transfection experiments, we found that over-expression of MYO1C significantly decreased cell proliferation, and silencing MYO1C with siRNA increased cell viability. Additionally, knockdown of MYO1C impaired the ability of cells to migrate, spread and adhere to the surface. Recent published studies suggested a potential interplay between MYO1C and the phosphoinositide 3-kinase (PI3K)/AKT pathway. To examine this hypothesis, we analyzed the expression and/or activation of components of the PI3K/AKT and RAS/ERK signaling pathways in vivo in CRC samples, and in vitro in cells transfected with the MYO1C gene expression construct or MYO1C-targeted siRNA. To identify other potential pathways/ mechanisms through which MYO1C may exert its tumor suppressor activity, we additionally performed new sets of MYO1C-siRNA knockdown experiments. At different time points post transfection, we performed microarray global gene expression experiments followed by bioinformatics analysis of the data. Altogether, the results suggested an early PI3K/AKT response to altered MYO1C expression. We additionally identified several cancer-related genes/pathways with late response to MYO1C knockdown. All things considered, the identification of MYO1C-expression impact on cell proliferation, migration, and adhesion in combination with its interplay between several cancer-related genes and signaling pathways provide further evidence for the initial hypothesis of a tumor suppressor activity of MYO1C

Shrimp Lipid Prevents Endoplasmic Reticulum-Mediated Endothelial Cell Damage

Shrimp contains a fat that benefits cardiovascular function and may help in the prevention of diseases. The stress of essential cellular organelle endoplasmic reticulum (ER) is linked to endothelial dysfunction and damage. This research aimed at investigating the effect of shrimp lipid (SL) on endothelial cells in response to ER stress, as well as the underlying mechanisms. Human endothelial cells were pretreated with SL (250 and 500 μg/mL) for 24 h, and treated with 0.16 μg/mL of Thapsigargin (Tg) for 24 h. The apoptosis and necrosis were detected by Hoechst 33342/propidium iodide (PI) co-staining. Cellular signaling pathways and ER stress markers were evaluated by Western blot analysis and immunofluorescence. SL protected against ER-induced endothelial cell apoptosis. According to the results, the viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1%, but SL (250 μg/mL) pretreatment increased cell viability to 77.26 ± 3.9%, and SL (500 μg/mL) increased to 72.42 ± 4.3%. SL suppressed the increase in ER stress regulator glucose-regulated protein 78 (GRP78) and attenuated the RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK) and inositol-requiring ER-to-nucleus signaling protein 1 (IRE1) pathways. SL could inhibit cell damage by reducing the ER-related apoptosis protein, C/EBPα-homologous protein (CHOP), induced by ER stress. Taken together, we found the protective effect and mechanism of SL in protecting ER stress-induced endothelial cell apoptosis through suppression of the ER stress pathway. The findings may support the potential use of SL as an approach with a protective effect on endothelial cells

Structural modification of resveratrol analogue exhibits anticancer activity against lung cancer stem cells via suppression of Akt signaling pathway

Abstract Background Compound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect. Methods Three human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFH2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein. Results In this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines (H23, H292, and A549). We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity. Conclusions Knowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer

Analysis of an independent tumor suppressor locus telomeric to Tp53 suggested Inpp5k and Myo1c as novel tumor suppressor gene candidates in this region

Several reports indicate a commonly deleted chromosomal region independent from, and distal to the TP53 locus in a variety of human tumors. In a previous study, we reported a similar finding in a rat tumor model for endometrial carcinoma (EC) and through developing a deletion map, narrowed the candidate region to 700 kb, harboring 19 genes. In the present work real-time qPCR analysis, Western blot, semi-quantitative qPCR, sequencing, promoter methylation analysis, and epigenetic gene expression restoration analyses (5-aza-2'-deoxycytidine and/or trichostatin A treatments) were used to analyze the 19 genes located within the candidate region in a panel of experimental tumors compared to control samples. RESULTS: Real-time qPCR analysis suggested Hic1 (hypermethylated in cancer 1), Inpp5k (inositol polyphosphate-5-phosphatase K; a.k.a. Skip, skeletal muscle and kidney enriched inositol phosphatase) and Myo1c (myosin 1c) as the best targets for the observed deletions. No mutation in coding sequences of these genes was detected, hence the observed low expression levels suggest a haploinsufficient mode of function for these potential tumor suppressor genes. Both Inpp5k and Myo1c were down regulated at mRNA and/or protein levels, which could be rescued in gene expression restoration assays. This could not be shown for Hic1. CONCLUSION: Innp5k and Myo1c were identified as the best targets for the deletions in the region. INPP5K and MYO1C are located adjacent to each other within the reported independent region of tumor suppressor activity located at chromosome arm 17p distal to TP53 in human tumors. There is no earlier report on the potential tumor suppressor activity of INPP5K and MYO1C, however, overlapping roles in phosphoinositide (PI) 3-kinase/Akt signaling, known to be vital for the cell growth and survival, are reported for both. Moreover, there are reports on tumor suppressor activity of other members of the gene families that INPP5K and MYO1C belong to. Functional significance of these two candidate tumor suppressor genes in cancerogenesis pathways remains to be investigated

Lowered Expression of Tumor Suppressor Candidate MYO1C Stimulates Cell Proliferation, Suppresses Cell Adhesion and Activates AKT.

Myosin-1C (MYO1C) is a tumor suppressor candidate located in a region of recurrent losses distal to TP53. Myo1c can tightly and specifically bind to PIP2, the substrate of Phosphoinositide 3-kinase (PI3K), and to Rictor, suggesting a role for MYO1C in the PI3K pathway. This study was designed to examine MYO1C expression status in a panel of well-stratified endometrial carcinomas as well as to assess the biological significance of MYO1C as a tumor suppressor in vitro. We found a significant correlation between the tumor stage and lowered expression of MYO1C in endometrial carcinoma samples. In cell transfection experiments, we found a negative correlation between MYO1C expression and cell proliferation, and MYO1C silencing resulted in diminished cell migration and adhesion. Cells expressing excess of MYO1C had low basal level of phosphorylated protein kinase B (PKB, a.k.a. AKT) and cells with knocked down MYO1C expression showed a quicker phosphorylated AKT (pAKT) response in reaction to serum stimulation. Taken together the present study gives further evidence for tumor suppressor activity of MYO1C and suggests MYO1C mediates its tumor suppressor function through inhibition of PI3K pathway and its involvement in loss of contact inhibition

List of primary antibodies and the optimized dilution.

<p>List of primary antibodies and the optimized dilution.</p