Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography–mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A2 (iPLA2) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA2 activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein–protein interacting partner of PRDX6 and HSPA9

Squalene loaded nanoparticles effectively protect hepatic AML12 cell lines against oxidative and endoplasmic reticulum stress in a TXNDC5-dependent way

This article belongs to the Special Issue Olive Oil Antioxidant.Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5.This research was supported by grants (CIBEROBN, CB06/03/1012, 1 January 2008) from CIBER Fisiopatología de la Obesidad y Nutrición as initiative of FEDER-ISCIII, Ministerio de Ciencia e Innovación-Fondo Europeo de Desarrollo Regional (PID2019-104915RB-I00, 1 June 2020) and Fondo Social Europeo-Gobierno de Aragón (B16_20R, 26 March 2020). S.H.B. was recipient of a joint fellowship from the Universities of Zaragoza and Pau and J.S.-M. was recipient of a Fundación Cuenca Villoro fellowship.Peer reviewe

Squalene Loaded Nanoparticles Effectively Protect Hepatic AML12 Cell Lines against Oxidative and Endoplasmic Reticulum Stress in a TXNDC5-Dependent Way

Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5

Expression Patterns of miR181a and miR30d in Patients with Breast Cancer

One of the important molecular pathways in breast cancer is the PTEN-PI3K-AKT pathway. Any change in the activity of the PTEN gene can alter the PI3K-AKT pathway. Moreover, there are subsets of genes and pathways their expression changes by post-transcriptional regulations. For instance, gene regulation alters by non-coding RNAs such as micro-RNAs as post-transcriptional regulators that prevent the expression of the target transcript. Therefore, it is essential to assess the related alterations in micro-RNA expression patterns to find out the possible causes of conversions in related transcripts and pathways such as the PTENPI3K-AKT pathway in breast cancer. To determine the expression level of miR-181a and miR-30d in 30 breast tumor samples and 30 adjacent normal samples, the RNA extraction, and cDNA synthesis was performed by RiboEx (GeneAll, Korea). Finally, the Real-Time PCR method was used for quantitative analysis of the expression levels of these miRNAs. all the experimental part of the project in done at Islamic Azad University in 2017. After analyzing comparisons in the expression level of miR-181a and miR-30d in tumor and normal tissues, there was a significant increase in the expression level of miR-181a in tumor samples compared with normal samples. Moreover, the expression level of miR-30d in tumor samples reported a significant decrease in comparison with normal samples (P <0.05). Upregulation of miR-181a may affect the transcription of the PTEN gene resulting in the cell progress to cancer. The Downregulation of miR-30d may also lead to cancer cell growth, due to a reduction in the affecting on the CREB gene transcript

Caracterización del elemento de respuesta al escualeno en el gen murino Cyp2b10

El gen CYP2B10 tiene un papel en el desarrollo de la enfermedad hepática, ya que metaboliza el etanol, y esto conduce a la generación de especies reactivas de oxígeno (ROS) con la capacidad de generar daño hepático que conduce a la enfermedad hepática. Otros estudios e investigaciones han encontrado que el escualeno tiene un papel protector en términos de la enfermedad hepática y que afecta la expresión de Cyp2b10. Por tanto, el objetivo de este estudio es la caracterización del promotor Cyp2b10, así como los efectos que tiene sobre él el escualeno. Para la realización de este estudio, el promotor se transfectará en una línea de AML12 (células hepáticas de ratón Alfa 12) y luego se tratará durante 48 horas con diferentes portadores de escualeno. Estos vehículos serán PLGA (poli (ácido láctico-co-glicólico)), gelicelina y etanol. También se utilizará otro componente, el lanosterol, como metabolito de escualeno para ver si el efecto se debe al escualeno o su metabolito. Como resultado de este estudio se ha encontrado que el escualeno reduce la expresión de Cyp2b10, que el mejor portador de los tres estudios es PLGA, seguido de gelicelina y luego etanol.<br /

KMT2D and IGF2 Genes Expression in Breast Cancer Patients

Background: Breast cancer which is often a cancer in women, is one of the most important public health problems and 1,384,155 new cases worldwide are associated with 459,000 deaths. Breast cancer is highly heterogeneous in its pathological characteristics. Current predictions and statistics suggest that both worldwide incidence of breast cancer and related mortality are on the rise. So far, many studies have been conducted on various genes in breast cancer that can help to treat this cancer. Methods: The gene expression of KMT2D and IGF2 were investigated in 35 samples with breast cancer after genomic RNA extraction and synthesis cDNA using quantitative real-time PCR method. Result: There was no significant difference in the expression of KMT2D and IGF2 genes in breast tumor samples compared to adjacent normal samples (P > 0.05). However, this study was designed as a pilot study, and further investigations are required to confirm our findings

Differentially Expressed Genes in Response to a Squalene-Supplemented Diet Are Accurate Discriminants of Porcine Non-Alcoholic Steatohepatitis

Squalene is the major unsaponifiable component of virgin olive oil, the fat source of the Mediterranean diet. To evaluate its effect on the hepatic transcriptome, RNA sequencing was carried out in two groups of male Large White x Landrace pigs developing nonalcoholic steatohepatitis by feeding them a high fat/cholesterol/fructose and methionine and choline-deficient steatotic diet or the same diet with 0.5% squalene. Hepatic lipids, squalene content, steatosis, activity (ballooning + inflammation), and SAF (steatosis + activity + fibrosis) scores were analyzed. Pigs receiving the latter diet showed hepatic squalene accumulation and twelve significantly differentially expressed hepatic genes (log2 fold change ENPEP) was correlated with triglyceride content, while alpha-fetoprotein (AFP), neutralized E3 ubiquitin protein ligase 3 (NEURL3), 2′-5′-oligoadenylate synthase-like protein (OASL), and protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) were correlated with activity reflecting inflammation and ballooning, and NEURL3 with the SAF score. AFP, ENPEP, and PPP1R1B exhibited a remarkably strong discriminant power compared to those pathological parameters in both experimental groups. Moreover, the expression of PPP1R1B, TMEM45B, AFP, and ENPEP followed the same pattern in vitro using human hepatoma (HEPG2) and mouse liver 12 (AML12) cell lines incubated with squalene, indicating a direct effect of squalene on these expressions. These findings suggest that squalene accumulated in the liver is able to modulate gene expression changes that may influence the progression of non-alcoholic steatohepatitis

Thioredoxin domain containing 5 is involved in the hepatic storage of squalene into lipid droplets in a sex-specific way

Hepatic thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family found associated with anti-steatotic properties of squalene and located in the endoplasmic reticulum and in lipid droplets. Considering that the latter are involved in hepatic squalene accumulation, the present research was aimed to investigate the role of TXNDC5 on hepatic squalene management in mice and in the AML12 hepatic cell line. Wild-type and TXNDC5-deficient (KO) mice were fed Western diets with or without 1% squalene supplementation for 6 weeks. In males, but not in females, absence of TXNDC5 blocked hepatic, but not duodenal, squalene accumulation. Hepatic lipid droplets were isolated and characterized using label-free LC-MS/MS analysis. TXNDC5 accumulated in this subcellular compartment of mice receiving squalene and was absent in TXNDC5-KO male mice. The latter mice were unable to store squalene in lipid droplets. CALR and APMAP were some of the proteins that responded to the squalene administration in all studied conditions. CALR and APMAP were positively associated with lipid droplets in the presence of squalene and they were decreased by the absence of TXNDC5. The increased squalene content was reproduced in vitro using AML12 cells incubated with squalene-loaded nanoparticles and this effect was not observed in an engineered cell line lacking TXNDC5. The phenomenon was also present when incubated in the presence of a squalene epoxidase inhibitor, suggesting a mechanism of squalene exocytosis involving CALR and APMAP. In conclusion, squalene accumulation in hepatic lipid droplets is sex-dependent on TXNDC5 that blocks its secretion